CN103773795A - Plants having increased yield-related traits and a method for making the same - Google Patents

Abstract

The present invention relates generally to the field of molecular biology and concerns a method for increasing various plant yield-related traits by increasing expression in a plant of a nucleic acid sequence encoding an ammonium transporter (AMT) polypeptide. The present invention also concerns plants having increased expression of a nucleic acid sequence encoding an AMT polypeptide, which plants have increased yield-related traits relative to control plants. The invention also provides constructs useful in the methods of the invention.

Description

There is plant and the production method thereof of the Correlated Yield Characters of enhancing

The application submits on November 21st, 2008, the divisional application of the PCT application PCT/EP2008/065947 that denomination of invention is " having plant and the production method thereof of the Correlated Yield Characters of enhancing ", it is on 05 24th, 2010 that described PCT application enters the date in China national stage, and application number is 200880117394.1.

The present invention relates generally to biology field, and relate to by plant improve coding ammonium transporter ( ammonium transporter, AMT) polypeptide nucleotide sequence expression and improve the method for various plants Correlated Yield Characters.The invention still further relates to the plant with the nucleotide sequence raising of expressing coding AMT polypeptide, described plant has the Correlated Yield Characters of raising compared with control plant.The present invention also provides the construct that can be used for the inventive method.

The world population of sustainable growth and agricultural have stimulated the research about increase farm efficiency with arable land supply atrophy.Conventional crop and the utilization of Horticulture improved means select breeding technique to identify the plant with welcome characteristic.But this type of selects breeding technique to have several defects, these technology generally expend a lot of work and produce such plant, and it often contains heterology hereditary component, and this may always not cause desired proterties to be transmitted from parental generation plant.Recent advances in molecular biology has allowed the mankind to improve the idioplasm of animal and plant.The genetic engineering of plant makes to separate and to operate genetic material (generally in DNA or rna form) and introduces subsequently this genetic material to plant.This type of technology has generation and possesses diversified economy, agronomy or the crop of Horticulture Ameliorative character or the ability of plant.

The proterties with special economic meaning is the output increasing.Output is normally defined measurable economic worth of making deposits yields.This can define with regard to quantity and/or quality aspect.Output directly depends on several factors, the number of such as organ and size, plant structure (for example number of branch), seed generation, leaf aging etc.Root development, nutrient intake, stress tolerance and early stage vigor (early vigor) can be also the important factors that determines output.Therefore, optimize aforementioned factor and can have contribution to increasing crop yield.

Seed production is the proterties of particularly important, and this is because the seed of many plants is most important for human and animal's nutrition.Account for the over half of total calorie of intake of the mankind such as corn, rice, wheat, rape (canola) and Soybean and Other Crops, no matter be the direct consumption by seed itself, still by the consumption of the meat products of being raised by the seed of processing.They are also the sources of industrial processes carbohydrate used, oils and multiclass metabolite.Seed contains embryo (new branch and the source of root) and endosperm (nutrition source of embryonic development in sprouting and seedling early growth process).The growth of seed relates to many genes, and needs metabolite to be transferred to the seed of growing from root, leaf and stem.Particularly endosperm, the metabolic precursor thereof of assimilation carbohydrate, oils and protein, is synthesized storage property polymer, to fill grain.

Phytomass is that fodder crop is as the output of clover, ensiling cereal and hay.In bread crop, use many alternate parameter of output.Wherein primary is estimation plant size.According to the difference of species and etap, can measure plant sizes by many methods, but comprise plant gross dry weight, dry weight, long-pending, the plant height of fresh weight, leaf area, caulome, lotus throne plant diameter, leaf length, root length, root biomass, tiller number and the number of sheets on the ground on the ground.Many species maintain the conservative ratio between plant different piece size in the given etap.Utilize these Allometric Relationships and these are carried out to extrapolation (as 2005Agric Ecosys & Environ105:213 such as Tittonell) from one to the other about big or small measuring result.The plant size of early development stage is conventionally by relevant with the plant size of etap in late period.The larger plant with larger leaf area can absorb more light and carbonic acid gas by smaller plant conventionally, therefore probably in the weightening finish same period more (Fasoula & Tollenaar 2005 Maydica50:39).Except the microenvironment that reaches at first size or prepotent potential continuity that plant has, this is its additive effect.Plant size and growth velocity exist strong hereditary component (as 2005Plant Physiology 139:1078 such as ter Steege), and probably relevant with the size under another kind of envrionment conditions (the 2003Theoretical Applied Genetics 107:679 such as Hittalmani) of the size of all diversified genotype plants under a kind of envrionment conditions up to now.By this way, use the alternate parameter of the diversified dynamic environment that standard environment meets with in different time and place as crop in field.

Early stage vigor for another important character of numerous crops.Improving early stage vigor is the important goal of modern rice breeding plan on temperate zone and tropical rice varieties.It is important for correct soil set that long root is planted in rice at water.In the situation that rice directly being sowed to flooded field, and in the situation that plant must emerge rapidly from water, longer seedling is relevant to vigor.In the situation that implementing drilling (drill-seeding), longer mesocotyl and coleoptile are important for well emerging.In artificial reconstructed plant, the ability of early stage vigor will be extremely important in agricultural.For example, corn (the Zea mayes L.) hybrid that bad early stage vigor has limited based on Corn Belt idioplasm (Corn Belt germplasm) is introduced a fine variety European Atlantic ocean region.

Harvest index is the ratio of seed production and ground dry weight, it is relatively stable under many envrionment conditionss, therefore between plant size and grain yield, conventionally can obtain more firm dependency (as 2002Crop Science42:739 such as Rebetzke).These methods link together inherently, because photosynthesis productivity 1985Physiology of Crop Plants.Iowa State University Press, pp68-73 such as () Gardener that most of cereal biomasss depend on leaf and stem is current or store.Therefore, to the selection of plant size, or even in the selection of growing commitment, be used as the index (as 2005 Agric Ecosys & Environ105:213 such as Tittonell) of following potential production.When test hereditary difference is during on the affecting of stress tolerance, greenhouse or plant culturing chamber have inherent advantages compared with field: can make operability and the light intensity stdn of soil function, temperature, water and nutrient.But, for want of wind-force or insect cause bad pollination, or because insufficient space is to allow matured root or canopy growth etc., the sex-limited meeting of these manual offices that output is caused limits these application controling environment in test volume variance.Therefore, under culturing room or greenhouse standard condition, measure the plant size of early development stage, be to provide the standard method of potential hereditary yield heterosis index.

Another important character is improved abiotic stress tolerance.Abiotic stress is the major cause of world wide Crop damage, reduces mean yield and exceed 50% (Wang etc., Planta (2003) 218:1-14) for most of staple crop plants.Abiotic stress can be caused by arid, salinity, extreme temperature, chemical toxicity, nutrient (macroelement and/or trace element) surplus or shortage, radiation and oxidative stress.Improving plant will have great economic advantages to peasant at world wide to the ability of abiotic stress tolerance and can allow during unfavourable condition and in arable farming otherwise be impossible land raise crop.

Crop yield thereby can increase by optimizing one of aforementioned factor.

Depend on end-use, may have precedence over other yield traits to the improvement of some yield traits.For example for application as feed or timber are produced or biofuel resource for, increasing phytoma part may wish, and for application as flour, starch or oil production, increase is planted a subparameter and may especially be wished.Even if in the middle of kind of subparameter, some parameter can be more preferably in other parameter, and this depends on application.Number of mechanisms can have contribution to increasing seed production, and no matter form is the seed size of increase or the number seeds of increase.

Increase a kind of method of Correlated Yield Characters (seed production and/or biomass) in plant and can be inherent growth mechanism by regulating plant as cell cycle or involved in plant growth or participate in the multi-signal approach of defense mechanism.

Have now found that and can in plant, express the multiple Correlated Yield Characters improving in plant by the nucleotide sequence that improves coding ammonium transporter (AMT) polypeptide in plant.The Correlated Yield Characters of described raising comprise following one or more: the root biomass of the early stage vigor of raising, the Aboveground Biomass of Young of raising, raising, every strain plant seed ultimate production of raising, the full rate of seed of raising, the full seed number of raising and the harvest index of raising.

Background

Ammonium and nitrate are the major nitrogen source of plant-growth and growth.Plant needs translocator to obtain ammonium and nitrate.The translocator of ammonium and nitrate does not exist only in plant, and is present in nearly all biology.Ammonium transporter (AMT) exists as gene family conventionally in genome, for example at least in Arabidopis thaliana (Arabidopsis thaliana), there are 6 kinds, in Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), there are 8 kinds (Gonzales-Ballester etc. (2004) Plant Molec Biol56:863-878), in poplar, there are 14 kinds (Couturier etc. (2007) New Phytologist174:137-150), in this diatom of Phaeodactylum tricornutum (Phaeodactylum tricornutum), there are 6 kinds (Allen (2005) J Phycology41).

Based on phylogenetic analysis, identify 3 subfamilies (Loqu é & von Wiren (2004) J Exp Bot55 (401): 1293-1305) of ammonium transporter:

1.AMT subfamily, comprises plant AMT1 type translocator and cyanobacteria ammonium transporter;

2.MEP subfamily, comprises plant AMT2 type translocator, yeast MEP translocator, intestinal bacteria AmtB and other protokaryon homologues;

3.Rh subfamily, only comprises humans and animals RH blood group antigen.

All AMT polypeptide are all highly hydrophobic membranins, have the transbilayer helix that at least 10 (common 11) infer.Many reports demonstration AMT polypeptide can be taken in ammonium in wide concentration range, but avidity is variant between different biologies.In some biology (as plant), high-affinity and low-affinity ammonium transporter (Gazzarini etc. (1999) Plant Cell 11:937-47) are identified.Except avidity characteristic, also identified other regulation mechanisms of ammonium picked-up, for example transcribe with transcriptional level on (Yuan etc. (2007) Plant Phys 143:732-744).

Use the corn ubiquitin promoter of constitutive expression, in two rice growing kinds (Taipei309 and Jarrah), cross the nucleotide sequence of having expressed from the coding AMT1 of rice.Compared with wild-type, the spray of transgenic line and the biomass of root occur declining seedling and early nutrition stage, while particularly cultivation under high ammonium nutrition (Hoque etc. (2006) Functional Plant Biol 33:153-163).Author's deduction, the transgenic plant in early days biomass decline of growth phase may be because because ammonium assimilation can not match and cause ammonium to accumulate in root with higher ammonium picked-up.

United States Patent (USP) 6,620,610 have described coding from the nucleotide sequence of the AMT1 polypeptide of Arabidopis thaliana, the carrier for expressing on yeast and bacterium of nucleotide sequence that comprises described coding AMT1.

United States Patent (USP) 6,833,492 have described the nucleotide sequence of coding from the AMT1 polypeptide of soybean, corn, wheat and rice.Describe coding AMT1 polypeptide or there is the nucleotide sequence of the AMT polypeptide of 90% amino acid sequence identity with the soybean AMT1 polypeptide that separates.Describe the Plants and Seeds of the recombinant nucleic acid sequence that comprises these peptide sequences of encoding, and produced the method for these plants.

Beyond thought, find now that the expression of the nucleotide sequence that improves coding AMT polypeptide gives the Correlated Yield Characters of plant raising compared with control plant.

According to an embodiment, the method that improves Correlated Yield Characters in plant compared with control plant is provided, be included in the expression that improves the nucleotide sequence of coding AMT polypeptide described herein in plant.Improve Correlated Yield Characters comprise following one or more: every inflorescence of the full rate of seed of the root biomass of the early stage vigor of raising, the Aboveground Biomass of Young of raising, raising, every strain plant seed ultimate production of raising, raising, the full seed number of raising, raising spend number and improve harvest index.

Definition

Polypeptides/proteins

Term " polypeptide " and " protein " are used interchangeably in this article, refer to the amino acid linking together by peptide bond in random length polymerized form.

Polynucleotide/nucleic acid/nucleotide sequence/nucleotide sequence

Term " polynucleotide ", " nucleotide sequence ", " nucleotide sequence ", " nucleic acid " are used interchangeably in this article and refer in random length polymerization without the Nucleotide in branch's form i.e. ribonucleotide or deoxyribonucleotide or these two combination.

Control plant

To select suitable control plant be the usual part that arranges of experiment and can comprise corresponding wild-type plant or without the corresponding plant of goal gene.Control plant is generally plant species or or even the identical kind identical with plant to be evaluated.Control plant can be also the inefficacy zygote of plant to be evaluated." control plant " not only refers to whole strain plant as used in this article, also refers to plant part, comprises seed and plants subdivision.

Homologue

" homologue " of protein comprises such peptide, oligopeptides, polypeptide, protein and enzyme, and they have amino acid substitution, disappearance and/or insertion and have similar biologic activity and functionally active to the non-modified protein that derives it with respect to the non-modified protein of discussing.

Disappearance refers to remove one or more amino acid from protein.

Insertion refers to the introducing in predetermined site in protein of one or more amino-acid residues.Insertion can comprise single or multiple amino acid whose aminoterminals fusions and/or carboxyl terminal merges and the interior insertion of sequence.Conventionally, less than aminoterminal fusion or carboxyl terminal fusion in the insertion meeting of aminoacid sequence inside, about 1-10 residue rank.The example of aminoterminal or carboxyl terminal fusion rotein or fusogenic peptide comprises as the binding domains of transcriptional activator used in yeast two-hybrid system or activation structure territory, bacteriophage coat protein, (Histidine)-6-label, glutathione S-transferase-label, albumin A, maltose binding protein, Tetrahydrofolate dehydrogenase, Tag100 epi-position, c-myc epi-position, FLAG

-epi-position, lacZ, CMP (calmodulin binding peptide), HA epi-position, PROTEIN C epi-position and VSV epi-position.

Replacement refers to the to have similar characteristics amino acid of other amino acid substitution protein of (as similar hydrophobicity, wetting ability, antigenicity, formation or destroy the tendency of α-helixstructure or beta sheet structure).Amino acid substitution is generally single residue, but can be a bunch collection property, and this depends on the functional constraint that is placed in polypeptide; Inserting can be about 1-10 amino-acid residue rank conventionally.Amino acid substitution preferably conservative amino acid is replaced.Conservative property substitution table is (seeing that for example Creighton (1984) Proteins W.H.Freeman and Company edits and following table 1) well-known in the art.

Table 1: the example that conservative amino acid is replaced

Residue	Conservative property is replaced	Residue	Conservative property is replaced
				Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln
				Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr
				Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp；Phe
				His	Asn；Gln	Val	Ile；Leu
Ile	Leu，Val	?	?

Amino acid substitution, disappearance and/or insert can use peptide synthetic technology well-known in the art as the solid phase method of peptide synthesis etc. or operated and easily carried out by recombinant DNA.Well-known in the art for operating DNA sequence dna with the method for producing protedogenous replacement, insertion or disappearance variant.For example, the technology that produces Substitution for the predetermined site place at DNA is well known to the skilled person and comprises M13 mutagenesis, T7-Gen vitro mutagenesis method (USB, Cleveland, OH), the site-directed mutagenesis (Stratagene of QuickChange, San Diego, CA), site-directed mutagenesis or other site-directed mutagenesis of PCR-mediation.

Derivative

" derivative " comprises such peptide, oligopeptides, polypeptide, wherein compared with the aminoacid sequence of the protein (as target protein matter) of natural existence form, the interpolation that they comprise the amino-acid residue that the amino-acid residue that exists with non-natural exists amino acid whose replacement or non-natural." derivative " of protein also comprises such peptide, oligopeptides, polypeptide; wherein, compared with the aminoacid sequence of the natural existence form of polypeptide, they comprise naturally occurring change (glycosylation, acidylate, isoprenylation, phosphorylation, myristoylation, sulfation etc.) amino-acid residue or non-natural change amino-acid residue.Compared with the aminoacid sequence of originating with derivative, this derivative can also comprise one or more non-aminoacid replacement base or the interpolation (for example reporter molecule or other part) of being covalently or non-covalently combined with described aminoacid sequence, as for promoting to detect the reporter molecule of this derivative combination, and the amino-acid residue existing with the non-natural that the aminoacid sequence of naturally occurring protein compares.

Straight homologues/paralog thing

Straight homologues and paralog thing comprise the evolution concept for describing gene ancestral relationship.Paralog thing is the gene that same species endogenous origin copies in ancestral gene, and straight homologues is from the different biological genes that originate from species formation, and also derives from common ancestral gene.

Structural domain

Term " structural domain " refers to along the sequence alignment result of evolution related protein and at one group of conservative amino acid of specific location.Although the amino acid in other position can change between homologue, but may be essential amino acid in the amino acid indication of the high conservative of specific location in structure, stability or the function aspects of protein.Structural domain is identified because of the conservative degree of the height by the aligned sequences of protein homology thing family, and they can be as identifying that thing is to determine whether the polypeptide of being discussed belongs to the peptide family of previously having identified arbitrarily.

Motif/consensus sequence/label

Term " motif " or " consensus sequence " or " label " refer to the short conserved regions in the sequence of evolution related protein.Motif is the high conservative part of structural domain often, but also can only comprise the part of structural domain, maybe can be positioned at (if whole amino acid of motif are positioned at outside the structural domain of definition) outside conserved domain.

Hybridization

Term " hybridization " is as defined herein the process that wherein complementary nucleotide sequence of homology is annealed each other substantially.Crossover process can be carried out completely in solution, and two kinds of complementary nucleic acid molecule are all in solution.Crossover process also can occur in the situation that one of complementary nucleic acid molecule is fixed to matrix as magnetic bead, agarose (Sepharose) pearl or any other resin.Crossover process also can one of complementary nucleic acid molecule be fixed to solid support as nitrocellulose filter or nylon membrane on or carry out be fixed on for example silicate glasses upholder (the latter is called nucleotide sequence array or microarray or is called nucleotide sequence chip) by for example photolithography in the situation that.For hybridization is occurred, conventionally by nucleic acid molecule thermally denature or chemical modification so that double-stranded unwinding become two strands and/or remove hair clip or other secondary structure from single stranded nucleic acid molecule.

Term " severity " refers to occur therein the condition of hybridization.The severity of hybridization is affected as temperature, salt concn, ionic strength and hybridization buffer form by condition.Conventionally, low stringency is chosen as in the time of definite ionic strength and pH lower than the hot melting temperature(Tm) (T of particular sequence _m) approximately 30 ℃.Medium stringency be now temperature lower than T _mapproximately 20 ℃, high stringency be now temperature lower than T _mapproximately 10 ℃.High stringency hybridization condition is generally used for and separates the hybridization sequences with target nucleic acid sequence with high sequence similarity.But nucleic acid molecule can depart from and because of the degeneracy of the genetic codon substantially the same polypeptide of still encoding in sequence.Thereby sometimes may need medium stringency hybridization condition to identify this type of nucleotide sequence molecule.

T _mbe under definite ionic strength and pH 50% target sequence with mate completely probe hybridization time temperature.T _mdepend on based composition and the length of solution condition and probe.For example, longer sequence hybridization specifically under comparatively high temps.From lower than T _mapproximately 16 ℃ until the 32 ℃ of maximum hybridization of acquisition speed.The existence of monovalent cation in solution reduced the Coulomb repulsion of two nucleotide sequence interchains, thereby promotes hybrid molecule to form; This effect is significantly (for greater concn, this effect can be ignored) for the na concn of 0.4M at the most.Methane amide reduces the melting temperature(Tm) of DNA-DNA and DNA-RNA duplex, and every percentage ratio methane amide reduces 0.6-0.7 ℃, and adds 50% methane amide and allow to hybridize at 30-45 ℃, although hybridization speed can reduce.Base-pair mismatch has reduced the thermostability of hybridization speed and duplex.On average and for large probe, every % base mispairing T _mdecline approximately 1 ℃.Depend on the type of hybrid molecule, T _mcan use following equation to calculate:

1) DNA-DNA hybrid molecule (Meinkoth and Wahl, Anal.Biochem., 138:267-284,1984):

T _m=81.5 ℃+16.6 × log ₁₀[Na ⁺] ^a+ 0.41 × %[G/C ^b] – 500 × [L ^c] ^-1– 0.61 × % methane amide

2) DNA-RNA or RNA-RNA hybrid molecule

T _m=79.8+18.5(log ₁₀[Na ⁺] ^a)+0.58(%G/C ^b)+11.8(%G/C ^b) ²-820/L ^c

3) few DNA or few RNA ^dhybrid molecule:

For <20 Nucleotide: T _m=2 (l _n)

For 35 Nucleotide: T of 20 – _m=22+1.46 (l _n)

^aor for other monovalent cation, but within the scope of 0.01 – 0.4M, be only accurate.

^bwithin the scope of 30%-75%, be only accurate for %GC.

^cthe length (in base pair) of L=duplex.

^doligo, oligonucleotide; Ln, useful length=2 × (G/C number)+(the A/T number) of primer.

Any non-specific binding of controlling that can numerous known technologies, for example with proteinaceous solution closed film, add heterology RNA, heterology DNA and SDS to hybridization buffer and with the processing of RNA enzyme.For non-homology probe, a series of hybridization can be undertaken by changing one of following condition: (i) reduce gradually annealing temperature (for example, from 68 ℃ to 42 ℃) or (ii) reduce gradually methane amide concentration (for example from 50% to 0%).Technician understands during hybridization can change and will maintain or change the many kinds of parameters of stringency.

Except hybridization conditions, hybridization specificity generally also depends on the function of post-hybridization washing.For removing because of the background due to non-specific hybridization, the salts solution washing of dilution for sample.The key factor of this type of washing comprises ionic strength and the temperature of final washing soln: salt concn is lower and wash temperature is higher, and the severity of washing is higher.Wash conditions is generally carried out in hybridization severity or lower than hybridization severity.Positive hybridization produces the signal that at least doubles background signal.Conventionally, described above for the suitable stringency of nucleic acid array hybridizing analytical method or gene amplification detection method.Also can select stricter or more undemanding condition.Technician understands during washing can change and will maintain or change the many kinds of parameters of stringency.

For example, the common high stringency hybridization condition that is greater than the DNA hybrid molecule of 50 Nucleotide for length is included in 65 ℃ hybridizes in 1 × SSC and 50% methane amide in 1 × SSC or at 42 ℃, washs subsequently at 65 ℃ in 0.3 × SSC.The example that is greater than the medium stringency hybridization condition of the DNA hybrid molecule of 50 Nucleotide for length is included in 55 ℃ hybridizes in 6 × SSC and 50% methane amide in 4 × SSC or at 40 ℃, washs subsequently at 50 ℃ in 2 × SSC.The length of hybrid molecule is the expection length of hybrid nucleic acid.In the time of the known making nucleic acid molecular hybridization of sequence, can and identify that by aligned sequences described conserved regions determine hybrid molecule length herein.1 × SSC is 0.15M NaCl and 15mM Trisodium Citrate; Hybridization solution and washing soln can comprise 5 × DenhardtShi reagent, 0.5-1.0%SDS, the fragmentation salmon sperm DNA of 100 μ g/ml sex change, 0.5% trisodium phosphate extraly.

In order to define the object of severity level, can be with reference to (2001) Molecular Cloning:a laboratory manual such as Sambrook, third edition Cold Spring Harbor Laboratory Press, CSH, New York or with reference to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and annual upgrade version).

Splice variant

As used in this article term " splice variant " comprise wherein excise, replace, be shifted or add selected intron and/or exon or wherein intron shortened or the variant of the nucleotide sequence that lengthens.This type of variant will be the bioactive a kind of variant that has wherein substantially retained protein; This can realize by the functional fragment of selective retention protein.This type of splice variant can find or can manually manufacture at occurring in nature.Well-known in the artly (to see for example Foissac and Schiex (2005), BMC Bioinformatics. for predicting with the method that separates this type of splice variant; 6:25).

Allelic variant

Allelotrope or allelic variant are the alternative forms of given gene, are positioned at identical chromosome position.Allelic variant comprises single nucleotide polymorphism (SNP) and little insertion/deletion (INDEL).The size of INDEL is less than 100bp conventionally.SNP and INDEL are formed on the maximum set of sequence variants in most of biological natural existence polymorphism strain.

Gene shuffling/orthogenesis

Consisting of of gene shuffling or orthogenesis: DNA reorganization repeatedly, suitably screening and/or selection have the improvement nucleic acid sequences to proteins of biologic activity or the variant of its part (Castle etc., (2004) Science 304 (5674): 1151-4 to produce coding subsequently; United States Patent (USP) 5,811,238 and 6,395,547).

Regulatory element/regulating and controlling sequence/promotor

Term " regulatory element ", " regulating and controlling sequence " and " promotor " are all used interchangeably in this article, and mean in a broad sense to realize the modulability nucleotide sequence of the sequence expression being attached thereto.Term " promotor " refers generally to be positioned at genetic transcription starting point upstream and participates in identification and in conjunction with RNA polymerase and other oroteins, thereby instructs the nucleotide sequence regulating and controlling sequence of the transcribed nucleic acid effectively connecting.Aforementioned term comprises from typical eukaryotic gene group gene and (comprises the TATA box required for accurate transcripting starting, tool is with or without CCAAT box sequence) in derivative transcriptional regulatory sequences and replying grow stimulation and/or outside stimulus or with tissue specificity mode change genetic expression additional adjustment element (as, upstream activating sequence, enhanser and silencer).This term also comprises the transcriptional regulatory sequences of typical prokaryotic gene, in the case it can Bao Kuo – 35 box sequences with/Huo – 10 box transcriptional regulatory sequences.Term " regulatory element " also comprises to be given, activates or strengthens synthetic fusion molecule or the derivative that nucleotide sequence molecule is expressed in cell, tissue or organ.

" plant promoter " comprises the regulatory element that mediation encoding sequence section is expressed in vegetable cell." plant promoter " preferred source is from vegetable cell, the plant that the nucleotide sequence treating to express in the inventive method and describe in this article of for example coming to use by oneself transforms.This is also applicable to other " plant " modulability signal, as " plant " terminator.Promotor for the nucleotide sequence upstream of the inventive method can be replaced by one or more Nucleotide, insert and/or disappearance and being modified, but do not disturb promotor, open reading frame (ORF) or 3 ' regulatory region be as terminator or functional or active away from other 3 ' regulatory region of ORF.The activity of promotor also likely because of modify the sequence of this promotor or by having more active promotor, even thoroughly replace this promotor from the promotor of allos biology and increase.For expressing in plant, as mentioned above, nucleotide sequence molecule must effectively be connected to or comprise suitable promotor, and wherein said promotor is on orthochronous point and with needed space expression pattern expressing gene.

In order to identify function equivalence promotor, can analyze promotor intensity and/or the expression pattern of candidate's promotor, for example, by this promotor being effectively connected with reporter gene and measuring expression level and the pattern of this report gene in various plants tissue.The suitable reporter gene of knowing comprises as β-glucuronidase or beta-galactosidase enzymes.Measure promoter activity by the enzymic activity of measuring β-glucuronidase or beta-galactosidase enzymes.Then can compare by promotor intensity and/or expression pattern and for example, with reference to promotor (for the inventive method).Or; can compare to measure promotor intensity by quantitative mRNA or by the mRNA level of nucleotide sequence used in the inventive method and housekeeping gene (as 18S rRNA) mRNA level; wherein use technology well known in the art; the Northern trace for example being undertaken by autoradiographic density analysis or quantitatively PCR in real time or RT-PCR (Heid etc., 1996 Genome Methods6:986-994).Conventionally, " weak promoter " refers to drive the promotor of encoding sequence low expression level." low-level " refers in each cell the level of the transcript of approximately 1/10000 the transcript transcript to approximately 1/100000 to approximately 1/5000000.On the contrary, " strong promoter " drives encoding sequence high level expression, or the level of the transcript of approximately 1/10 the transcript transcript to approximately 1/100 to approximately 1/1000 in each cell.Conventionally, " medium tenacity promotor " refers to such promotor, and it all drives encoding sequence to express lower than the level of 35S CaMV promoter regulation in all cases.

Effectively connect

Term " effectively connection " refers to functionally be connected between promoter sequence and goal gene as used in this article, transcribes to such an extent as to promoter sequence can start goal gene.

Constitutive promoter

" constitutive promoter " refers at least one cell, tissue or organ in its great majority (but not necessarily whole) g and D stage and under most of envrionment conditionss, has the promotor of transcriptional activity.Following table 2a has provided the example of constitutive promoter.

Table 2a: the example of plant constitutive promoter

All in promotor

All over substantially all having activity in tissue or cell in promotor at biology.

Grow modulability promotor

Grow modulability promotor and having activity during certain growth period or in experience is grown the plant part changing.

Inducible promoter

Replying chemical, (summary is shown in Gatz 1997 to inducible promoter, Annu.Rev.Plant Physiol.Plant Mol.Biol., the transcripting starting that 48:89-108), there is induced or increase when environmental stimulus or physical stimulation, can be maybe " stress induced ", in the time that being exposed to various abiotic stress condition, plant activated, or " pathogen-inducible ", in the time that being exposed to multiple pathogens, plant activated.

Organ specificity/tissue-specific promoter

Organ specificity or tissue-specific promoter can be preferentially start the promotor of transcribing in some organ or tissue in as leaf, root, seed tissue etc.For example, " root-specific promoter " is promotor advantage in roots of plants with transcriptional activity, and essentially no activity in any other parts of plant, although allow any leakage to express in these other parts of plant.Can only in some cell, start the promotor of transcribing and be called in this article " cell-specific ".

The example of the root-specific promoter 2b that is listed in the table below:

Table 2b: the example of root-specific promoter

Seed specific promoters mainly has transcriptional activity in seed tissue, but not necessarily only in seed tissue, has (leaking situation about expressing).Seed specific promoters can have activity in seed development and/or germination process.The example of seed specific promoters is shown in below shows 2c.Other examples of seed specific promoters provide in Qing Qu and Takaiwa (Plant Biotechnol.J.2,113-125,2004), and its disclosure is incorporated herein by reference by entirety.

Table 2c: the example of seed specific promoters

Chlorenchyma specificity promoter is as defined herein the promotor mainly in chlorenchyma with transcriptional activity, and essentially no activity in any other parts of plant, although allow any leakage to express in these other parts of plant.

The example of the chlorenchyma specificity promoter that can be used for implementing the inventive method shows in following table 2d.

Table 2d: the example that chlorenchyma specificity starts

Another example of tissue-specific promoter is meristematic tissue specificity promoter, it mainly has transcriptional activity in merism tissue, essentially no activity in any other parts of plant, although allow any leakage to express in these other parts of plant.The example that can be used for the meristematic tissue specificity promoter of implementing the inventive method is shown in below shows 2e.

Table 2e: the example of meristematic tissue specificity promoter

Terminator

Term " terminator " comprises such regulating and controlling sequence, and it is the DNA sequence dna at transcription unit's end, sends primary transcript is carried out to the signal that 3 ' processing poly-adenosine and termination are transcribed.Terminator can be from natural gene, from multiple other plant gene or from T-DNA.Terminator to be added can be from for example nopaline synthase or octopine synthase gene, or from other plant gene or more preferably from any other eukaryotic gene.

Regulate

Term " adjusting " with regard to expression or genetic expression, mean such process, wherein expression level expression because of described gene compared with control plant changes, and preferably, expression level increases.Original not modulated expression can be that any type of structure RNA (rRNA, tRNA) or mRNA is expressed, and is translation subsequently.Term " adjusting is active " should mean any variation of nucleotide sequence of the present invention or coded protein expression, and this causes the output of plant increase and/or the growth of increase.

Expression/the mistake increasing is expressed

Term as used in this article " expression that increases " or " cross express " to mean for original wild-type expression level be that extra any form is expressed.

In this area, record in detail for increasing the method for gene or gene product expression and they and for example comprised, expressed, used transcriptional enhancer or translational enhancer by crossing of suitable promoters driven.Separated nucleic acid sequence as promotor or enhancer element can be imported in the suitable location of the polynucleotide of non-allos form (being generally upstream), so that the expression of the nucleotide sequence of upper tone coded desired polypeptides.For example, internal promoter can be changed in vivo and (be seen Kmiec, US 5,565,350 by sudden change, disappearance and/or displacement; Zarling etc., WO9322443), maybe the promotor separating can be imported to vegetable cell with the correct direction with respect to gene of the present invention and distance, so that controlling gene is expressed.

If desired expression of polypeptides, wishes to comprise poly-adenosine district at the 3 ' end in polynucleotide encoding district conventionally.Poly-adenosine district can be from natural gene, from multiple other plant gene or from T-DNA.3 ' end sequence to be added can be from for example nopaline synthase or octopine synthase gene or alternatively from another plant gene or more not preferably from any other eukaryotic gene.

Intron sequences also can be added on the encoding sequence of 5 ' non-translational region (UTR) or part coding property sequence, to be increased in the amount of the ripe information accumulating in cytosol.Verified can montage intron being included in plant expression constructs and animal expression construct transcription unit on mRNA level and protein level, increase genetic expression to 1000 times of (Buchman and Berg (1988) Mol.Cell biol.8:4395-4405 nearly; Callis etc. (1987) Gens Dev1:1183-1200).This type of intron enhancement of genetic expression is the strongest generally near being positioned at transcription unit's 5 ' end time.It is known in the art using corn intron A dh1- S introne 1,2 and 6, Bronze-1 intron.For general information, see: " corn handbook ", the 116th chapter, editor Freeling and Walbot, Springer, N.Y. (1994).

Native gene

" endogenous " mentioned in this article gene not only refers to the gene of being discussed existing with its natural form (without any the mankind intervene) as in plant, also refers to the homologous genes (or substantially nucleic acid/the gene of homology) of (again) the subsequently importing plant (transgenosis) in unpack format.For example, contain this genetically modified transgenic plant and can meet with the significantly reduction that transgene expression significantly reduces and/or native gene is expressed.

The expression reducing

The expression of " expression of reduction " mentioned in this article or " reduce or substantially remove " means native gene expression and/or polypeptide level and/or the polypeptide active reduction with respect to control plant.Compared with control plant, reduce or substantially to remove be at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90% to increase progressively preferred sequence, or 95%, 96%, 97%, 98%, 99% or more reduction.

In order to reduce or substantially to remove the expression of native gene in plant, need the continuous Nucleotide substantially of the sufficient length of nucleotide sequence.In order to carry out gene silencing, this length can be few to 20,19,18,17,16,15,14,13,12,11,10 or Nucleotide still less, or this length can the whole gene of as many as (comprising 5 ' and/or 3 ' UTR, part or all).Substantially continuous nucleotide fragments can come the nucleotide sequence (target gene) of own coding target protein matter or any nucleotide sequence from straight homologues, paralog thing or the homologue of the target protein matter of can encoding.Preferably, substantially continuous nucleotide fragments can form hydrogen bond with target gene (sense strand or antisense strand), more preferably, continuous nucleotide fragments has 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to increase progressively preferred sequence and target gene (sense strand or antisense strand) substantially.Coding (functional) polypeptide nucleotide sequence be not discussed herein for reducing or substantially remove native gene express several different methods required.

This reduction of expressing or basic removal can be used conventional tools and techniques to complete.For reducing or substantially to remove a kind of method that native gene expresses be to use the silence that mediates by RNA, wherein use nucleotide sequence or its part (be in the case from goal gene or from any nucleotide sequence derivative one section of continuous nucleotide sequence substantially, wherein said any nucleic acid can encode straight homologues, paralog thing or the homologue of target protein matter) inverted repeats (preferably can form hairpin structure).Another example of RNA silencing methods comprise with sense orientation import nucleotide sequence or its part (be in the case from goal gene or from any nucleic acid derivative one section of continuous Nucleotide substantially, wherein said any nucleic acid can encode straight homologues, paralog thing or the homologue of target protein matter) to plant.Another example of RNA silencing methods comprises use anti sense nucleotide sequence.Gene silencing also can be for example, by insertion mutagenesis (T-DNA inserts or transposon inserts) or by realizing as the strategy of Angell and Baulcombe (J.20 (3): 357-62 of (1999) Plant), (Amplicon VIGS WO 98/36083) or Baulcombe (WO 99/15682) and other people description.Other method, as used for the antibody of endogenous polypeptide to suppress the function of this polypeptide in plant, or the signal pathway that disturbs described polypeptide to participate in, will be well-known for technician.Artificial and/or natural microRNA (miRNA) can be used for knocking out genetic expression and/or mRNA translation.Endogenous miRNA is the little RNA of strand of a common 19-24 length of nucleotides.The artificial microRNA (amiRNA) of common 21 length of nucleotides can genetic modification with the genetic expression of the single or multiple goal gene of negative regulator specifically.The determinative of the selection of plant micrornas target is well-known in the art.Determine and can be used for the specific amiRNA of aided design (Schwab etc., (2005) Dev Cell8 (4): 517-27) for the empirical parameter of target identification.Also be the public obtainable (Schwab etc., (2006) Plant Cell18 (5): 1121-33) for the convenient tool that designs and produce amiRNA and precursor thereof.

For optimum performance, the gene silent technology of expressing in plant for reducing native gene need to use from monocotyledonous nucleotide sequence with transforming monocots, and uses nucleotide sequence from dicotyledons to transform dicotyledons.Preferably, will import in same species from the nucleotide sequence of any given plant species.For example, the nucleotide sequence from rice is converted into rice plant.But, the identical plant species of plant that not definitely requires nucleotide sequence to be imported to originate from will to import with this nucleotide sequence.As long as exist sizable homology just enough between endogenous target gene and nucleotide sequence to be imported.

Above-described be for reducing or substantially remove the example of the several different methods that native gene expresses in plant.Those skilled in the art can adjust aforementioned for reticent method to such an extent as to for example by utilizing suitable promotor realize whole strain plant or reduce the expression of native gene in its part easily.

Selective marker (gene)/reporter gene

" selective marker ", " selectable marker gene " or " reporter gene " comprise any gene from phenotype to cell that give, wherein identify and/or select the cell with nucleotide sequence construct institute's transfection of the present invention or conversion with promotion at gene described in described cell inner expression.These marker gene can be identified by a series of different principle the successful transfer of nucleotide sequence molecule.Suitable mark can be selected from the mark of giving antibiotics resistance or Herbicid resistant, the new metabolism proterties of introducing or allowing visual selection.The example of selectable marker gene comprise give antibiotics resistance gene (as make the nptII of Liu Suanyan NEOMYCIN SULPHATE and kantlex phosphorylation or make the hpt of Totomycin phosphorylation or give to for example bleomycin, Streptomycin sulphate, tsiklomitsin, paraxin, penbritin, gentamicin, Geneticin (Geneticin) (G418), the gene of the resistance of spectinomycin or blasticidin), the gene of conferring herbicide resistance (for example provides Basta

the bar of resistance; AroA or the gox of glyphosate resistance is provided or gives for example gene of the resistance of imidazolone, phosphinothricin or sulfourea) or provide metabolism proterties gene (as allow plant use seminose as the manA of sole carbon source utilize the xylose isomerase of wood sugar or anti-nutrition mark as 1,5-anhydroglucitol resistance).The expression of visual marker gene causes forming color (for example such as X-Gal of β-glucuronidase, GUS or beta-galactosidase enzymes substrate coloured with it), luminous (as luciferin/luciferase system) or entangles light (green is entangled photoprotein GFP and derivative thereof).This list only represents the possible mark of minority.Technician is familiar with this type of mark.Depend on biology and system of selection, preferably different marks.

Known to nucleotide sequence is stable or integration,temporal during to vegetable cell, the only cellular uptake foreign DNA of small portion and be integrated into as required cellular genome, this depends on the rotaring dyeing technology of expression carrier used thereof and use.For identifying and select these intasomies, conventionally the gene of codes selection mark (one of as described above) is introduced to host cell together with goal gene.These marks therein these genes because using in the non-functional mutant of disappearance due to ordinary method for example.In addition, the nucleotide sequence molecule of codes selection mark can be introduced in host cell, its with the sequence that comprises polypeptide used in code book invention polypeptide or the inventive method in identical carrier, or on independent carrier.Can be for example identify (for example thering is the cell survival of selective marker of integration and other necrocytosis) by selection with the cell of the nucleotide sequence stable transfection of introducing.

Once because successfully introduced nucleotide sequence, in genetically modified host cell, just no longer need or do not wish marker gene, especially antibiotics resistance gene and herbicide resistance gene, therefore advantageously uses for introducing the inventive method of nucleotide sequence the technology that can remove or excise these marker gene.A kind of be called cotransformation method as this method.Cotransformation method is used two kinds of carriers for transforming simultaneously, and a kind of carrier carries nucleotide sequence of the present invention and another kind of carrier carries marker gene.A high proportion of transformant is accepted, or the in the situation that of plant, comprise (up to 40% or more transformant) these two kinds of carriers.In the situation that using Agrobacterium-mediated Transformation, transformant is only accepted a part for carrier conventionally, and flank has the sequence of T-DNA, and it represents expression cassette conventionally.Marker gene can be removed by hybridizing subsequently from the plant transforming.In another approach, the marker gene that is integrated into transposon is used for transforming (being called Ac/Ds technology) together with the nucleotide sequence of wanting.Transformant can be instantaneous or stably transform with the nucleotide sequence construct that causes transposase to be expressed with originate plant hybridization or transformant of transposase.(about 10%) in some cases, transposon successfully occurs while conversion, jump out the genome of host cell and lose.Under other more susceptible condition, transposon skips to different positions.In these cases, marker gene must be removed by hybridizing.In microbiology, develop the technology that realizes or promote to detect this class event.Another favourable method depends on known recombination system; The advantage of this method is to remove by hybridization.The most well-known system of the type is called Cre/lox system.Cre1 is the recombinase that removes sequence between loxP sequence.If marker gene is integrated between loxP sequence, once conversion successfully occurs, expresses and remove marker gene by recombinase.Other recombination system is HIN/HIX, FLP/FRT and REP/STB system (Tribble etc., J.Biol.Chem., 275,2000:22255-22267; Velmurugan etc., J.Cell Biol., 149,2000:553-566).Likely nucleotide sequence of the present invention is integrated into Plant Genome in locus specificity mode.These methods also can be applied to microorganism naturally as yeast, fungi or bacterium.

Genetically modified/transgenosis/restructuring

For the object of the invention, " genetically modified ", " transgenosis " or " restructuring " mean expression cassette, gene construct or the carrier that comprises this nucleotide sequence or the biology transforming with nucleotide sequence of the present invention, expression cassette or carrier with regard to nucleotide sequence for example, all these structures all produce by recombination method, wherein

(a) coding is used for the nucleic acid sequences to proteins of the inventive method, or

(b) the genetic regulation sequence being effectively connected with nucleotide sequence of the present invention, for example promotor, or

(c) a) and b)

Not in its natural genotypic environment or modified by genetic manipulation method, be modified with may for example adopt replace, add, disappearance, inversion or insert the form of one or more nucleotide residues.Natural genotypic environment is interpreted as natural gene group locus or the chromogene seat that means to originate in plant or exists in genomic library.The in the situation that of genomic library, the natural genotypic environment of nucleotide sequence is preferably retained, and is retained at least in part.This environment is distributed at least one side of nucleotide sequence and has at least 50bp, preferably 500bp at least, particularly preferably 1000bp at least, the most preferably sequence length of 5000bp at least.The natural promoter of for example nucleotide sequence of naturally occurring Biao Da He – and the naturally occurring combination of the corresponding nucleotide sequence of polypeptide used in code book inventive method, Ru above Suo is Dinged Yi – in the time that this expression cassette is subject to modifying by non-natural synthetic (" manually ") method (as mutagenic treatment), becomes transgene expression cassette.Appropriate method is for example at US 5,565,350 or WO 00/15815 in describe.

For the object of the invention, therefore transgenic plant are as above interpreted as the natural gene seat that means nucleotide sequence used in the inventive method and be not arranged in described this nucleic acid of Plant Genome, and described nucleotide sequence likely homology or allos ground is expressed.But as mentioned, although transgenosis also means nucleotide sequence of the present invention or in the methods of the invention in the natural place of nucleotide sequence used this nucleic acid in Plant Genome, but its sequence modified for native sequences, and/or the adjusting sequence of described native sequences is modified.Transgenosis is preferably interpreted as and means to express in the non-natural locus of nucleotide sequence of the present invention in genome, and homology expression or the preferred heterogenous expression of nucleotide sequence occur.Preferred transgenic plant are for mentioning herein.

Transform

Term " introducing " or " conversion " comprise exogenous polynucleotide are transferred in host cell as mentioned in this article, no matter what are for the method transforming.Can be follow-up the plant tissue of clone's property propagation (no matter occur by organ or embryo occurs) can transform and the whole strain plant that can therefrom regenerate with genetic constructs of the present invention.The concrete tissue of selecting will depend on clone's property proliferating system of the concrete species that can be used for and be suitable for just transforming most.Example organization target comprises the meristematic tissue (for example cotyledon meristematic tissue and hypocotyl meristematic tissue) of leaf dish, pollen, embryo, cotyledon, hypocotyl, megagametophyte, callus, existing meristematic tissue (for example apical meristem, axillalry bud and root meristematic tissue) and induction.Polynucleotide can instantaneous or stably be introduced host cell and can maintain to nonconformity, for example, as plasmid.Or polynucleotide can be integrated in host genome.The transformed plant cells producing can be used for regenerating in the manner known to persons skilled in the art conversion of plant subsequently.

Alien gene is converted into and in Plant Genome, is called conversion.The conversion of plant species is quite conventional technology now.Advantageously, the either method in several method for transformation can be used for goal gene to introduce suitable ancester cell.For from plant tissue or vegetable cell transforms and the described method of the plant that regenerates can be for instantaneous conversion or for stable conversion.Method for transformation comprise the chemical that uses liposome, electroporation, increase dissociative DNA to take in, DNA direct injection to plant, particle gun blast technique, use conversion method and the microinjection of virus or pollen.Method for transformation can be selected from calcium/polyoxyethylene glycol method (Krens, F.A. etc., (1982) Nature296, the 72-74 for protoplastis; (1987) Plant Mol Biol8:363-373 such as Negrutiu I); The electroporation ((1985) Bio/Technol3, the 1099-1102 such as Shillito R.D.) of protoplastis; Microinjection (Crossway A etc., (1986) Mol.Gen Genet202:179-185) to vegetable material; Be coated with Particle bombardment (Klein TM etc., (1987) Nature327:70), (nonconformity) virus infection method etc. of DNA or RNA.Transgenic plant, comprise genetically modified crops plant, preferably produce by agriculture bacillus mediated conversion method.Favourable method for transformation is the conversion method of in plant (in planta).For this purpose, for example likely make Agrobacterium act on plant seed or likely with the meristematic tissue of Agrobacterium inoculation plant.Verifiedly according to the present invention make the Agrobacterium suspension that transforms to act on complete plant or at least act on flower primordium be particularly advantageous.Plant continues to cultivate until obtain the seed (Clough and Bent, Plant J. (1998) 16,735 – 743) of the plant of processing subsequently.The method transforming for agriculture bacillus mediated rice comprises the known method transforming for rice, those methods as described in arbitrary following document: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199:612-617,1996); (the Plant Mol Biol22 (3): 491-506 such as Chan, 1993), Hiei etc. (Plant J6 (2): 271-282,1994), its disclosure is incorporated herein by reference in this article, as provided completely.In the situation that corn transforms, preferred method is as (Nat.Biotechnol 14 (6): 745-50 such as Ishida, 1996) or (the Plant Physiol 129 (1): 13-22 such as Frame, 2002) describe, its disclosure is incorporated herein by reference in this article as fully.Described method by way of example mode further by B.Jenes etc., Techniques for Gene Transfer, come from Transgenic Plants, the 1st volume, Engineering and Utilization, editor S.D.Kung and R.Wu, Academic Press (1993) 128-143 and at Potrykus Annu.Rev.Plant Physiol.Plant Molec.Biol.42 (1991) 205-225) in describe.Nucleotide sequence to be expressed or construct are preferably cloned into the carrier that is suitable for transforming agrobacterium tumefaciens (Agrobacterium tumefaciens), such as pBin19 (Bevan etc., Nucl.Acids Res.12 (1984) 8711).The Agrobacterium being transformed by this carrier subsequently can be according to known way for conversion of plant, the plant for example using as model, as Arabidopis thaliana, (Arabidopsis is in scope of the present invention, be not considered as crop plants) or crop plants, for example tobacco plant, by soaking the leaf of abrasive leaf or chopping and subsequently they being cultivated and transformed in suitable substratum in Agrobacterium solution.The conversion of plant by agrobacterium tumefaciens for example by

vectors for Gene Transfer in Higher Plants, is described in 9877 or especially from F.F.White at Nucl.Acid Res. (1988) 16 with Willmitzer; At Transgenic Plants, the 1st volume, Engineering and Utilization, editor S.D.Kung and R.Wu, Academic Press, knows in 1993, the 15-38 pages.

Except transformant cell (its subsequently must the complete plant of regeneration), the also likely merismatic cell of conversion of plant and special those cells that develop into gamete that transform.In this case, the gamete of conversion is followed natural development of plants process, produces transgenic plant.Therefore, for example Arabidopis thaliana seed is processed with Agrobacterium and obtain seed from is grown plant, wherein a certain proportion of described plant is transformed and is therefore genetically modified [Feldman, KA and Marks MD (1987) Mol Gen Genet208:274-289; Feldmann K (1992).Come from C Koncz, N-H Chua and J Shell write, Methods in Arabidopsis Research, Word Scientific, Singapore, 274-289 page].Alternative method is based on repeatedly removing inflorescence and making in rosette excision position in the heart and the Agrobacterium of conversion hatches, thereby the seed transforming can obtain at more late time point equally, and (Chang (1994) Plant J.5:551-558; Katavic (1994) Mol Gen Genet, 245:363-370).But especially effective means is the vacuum infiltration method of improvement, as " flower is contaminated " method.The in the situation that of Arabidopis thaliana vacuum infiltration method, complete plant is under reduced pressure used the processing of Agrobacterium suspension [Bechthold, N (1993).C R Acad Sci Paris Life Sci, 316:1194-1199], and " flower dip method " in the situation that, the flower tissue of growing and the of short duration [Clough of hatching of Agrobacterium suspension of tensio-active agent processing, SJ and Bent, AF (1998) The Plant J.16,735-743].Gathered in the crops in both cases a certain proportion of transgenic seed, and these seeds can be distinguished with non-transgenic seed by cultivating under selection condition as above.In addition, the stable conversion of plastid is favourable because plastid in most of crop with the heredity of parent mode, reduce or eliminated transgenosis through pollen flow risk.The conversion of chloroplast gene group generally by Klaus etc., 2004[Nature Biotechnology22 (2), 225-229] in the exemplary method realization of being shown.In brief, sequence to be transformed is cloned into together with selectable marker gene between the flanking sequence of chloroplast gene group homology.The flanking sequence of these homologies instructs locus specificity to be integrated in plastom.Numerous different plant species are described plastid transformation and summarized and can come from Bock (2001) transgenosis plastid (Transgenic plastids in basic research and plant biotechnology) .J Mol Biol.2001 September 21 in fundamental research and Plant Biotechnology; 312 (3): 425-38 or Maliga, P (2003) plastid transformation technology commercialization progress (Progress towards commercialization of plastid transformation technology) .Trends Biotechnol.21,20-28.Further biotechnology progress has been made report with the form of unmarked plastid transformation body recently, described unmarked plastid transformation body can produce (Klaus etc. by the instantaneous marker gene of integrating altogether, 2004, Nature Biotechnology22 (2), 225-229).

T-DNA activates label

T-DNA activates label Science (1992) 1350-1353 such as () Hayashi and relates in the genome area of goal gene or upstream, gene coding region or downstream 10kb sentence structure like this and insert T-DNA (conventionally containing promotor (can be also translational enhancer or intron)), makes promotor instruct the expression of being determined gene by target.Conventionally the regulating effect that the natural promoter of, determining gene by target is determined genetic expression to described target is destroyed and this gene is under the promotor control of new introducing.Promotor is generally embedded in T-DNA.This T-DNA inserts Plant Genome randomly, for example, by agroinfection, and causes near the modification of the gene inserted T-DNA to be expressed.Because the modification of the gene near the promotor of introducing is expressed, the transgenic plant performance dominant phenotype of generation.

TILLING

Term TILLING is the abbreviation of " local damage of genome interior orientation induction ", refers to for generation of and/or identify the induced-mutation technique of nucleotide sequence, and wherein said nucleic acid sequence encoding has to modify expresses and/or active protein.The plant that TILLING also allows selection to carry this type of mutation variants.These mutation variants may be displayed on aspect, Huo position, intensity aspect or the expression (if for example sudden change affects promotor) in modification aspect the time.These mutation variants can show than by showed active higher activity in the gene of its natural form.TILLING is by high-density mutagenesis and high-throughput screening method combination.The general step of following in TILLING is: (Redei GP and Koncz C (1992) are at Methods in Arabidopsis Research in (a) EMS mutagenesis, Koncz C, Chua NH, Schell J edits, Singapore, World Scientific Publishing Co, the 16th 82 pages of –; Feldmann etc., (1994), at Meyerowitz EM, Somerville CR edits, Arabidopsis.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 137-172 page; Lightner J and Caspar T (1998) be at J Martinez-Zapater, J Salinas editor, Methods on Molecular Biology the 82nd volume, Humana Press, Totowa, NJ, 91-104 page); (b) individual DNA prepares and collects; (c) pcr amplification object district; (d) sex change and annealing are to allow to form heteroduplex; (e) DHPLC, is wherein detected as an extra peak in color atlas collecting the heteroduplex existing in thing; (f) identify mutated individual; (g) to the order-checking of sudden change PCR product.Method for TILLING is (McCallum etc., (2000) Nat Biotechnol 18:455-457 well-known in the art; Summary is shown in Stemple (2004) Nat Rev Genet5 (2): 145-50).

Homologous recombination

Homologous recombination allows the nucleotide sequence of selecting in the selected position of determining, to introduce in genome.Homologous recombination be in bio-science routinely for unicellular lower eukaryote as the standard technique of yeast or liver moss sword-like leave moss (Physcomitrella).The method that is used for carrying out homologous recombination plant is not only to model plant (Offringa etc. (1990) EMBO J9 (10): 3077-84) but also to such as rice of crop plants (Terada etc. (2002) Nat Biotech20 (10): 1030-4; Iida and Terada (2004) Curr Opin Biotech15 (2): 132-8) be described.

Output

What term " output " meant economic worth conventionally can measuring result, general with specify crop, and area relevant with the time period.Number, size and/or the weight of single plant part based on them and directly output is had to contribution, or actual output is the every acre yield for certain crop and a year, this determines divided by the acreage of plantation by ultimate production (comprise results with the output of evaluating)." output " of term plant can be for example, with nourishing body biomass, organ of multiplication and/or the propagulum of this plant (seed) relevant.

Early stage vigor

" early stage vigor " refer to enliven, healthy, well balanced growth (particularly during plant-growth is early stage), and can produce because plant fitness increases, its reason is that for example plant adapts to its environment (optimizing the distribution between use and the Miao Yugen of the energy) better.The plant with early stage vigor also shows the seedling survival of increase and better crop foundation, this often causes highly field (crop fitly grows, and most plants reaches each stage of growth on the substantially the same time) uniformly and better and higher output often.Thereby early stage vigor can be determined as thousand seed weight, germination percentage, the percentage ratio of emerging, growth of seedling, seedling height, root length, root and seedling biomass and numerous other factors by the multiple factor.

Increase/improve/strengthen

Term " increase ", " improvement " or " enhancing " is interchangeable and should in the application's implication, refers at least 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more output and/or growth compared with control plant as defined herein.

Seed production

The seed production itself increasing can show as following one or more indexs: a) seed biomass (seed gross weight) increases, and this can be based on single seed and/or every strain plant and/or per hectare or every acre; What b) each inflorescence and/or every strain plant increased spends number; C) (full) seed number increasing; D) the full rate of seed (it is expressed as the ratio between full seed number and seed sum) increasing; E) harvest index increasing, it is expressed as the ratio that can gather in the crops part (as seed) output and total biomass; And f) primary panicles (primary panicles) number that increases; G) thousand seed weight (TKW) increasing, this full seed number from counting and gross weight extrapolation thereof.The TKW increasing can be because of due to the seed size and/or seed weight that increase, and also can be because of due to the increase of embryo and/or endosperm size.

The increase of seed production also can show as the increase of seed size and/or seed volume.In addition, the increase of output itself can self-expression be also the increase of seed area and/or seed length and/or seed width and/or seed girth.The seed production increasing also can produce the structure of improvement, or can occur because of the structure of improvement.

Green degree index

" green degree index " calculates according to the digital picture of plant as used herein.For each pixel that belongs to plant target in image, calculate the ratio of green value with respect to red value (at the RGB model for encoded colors).Green degree index is expressed as the green red pixel per-cent than exceeding given threshold value.Under the growth conditions declining under normal growth condition, under salt stress growth conditions, at nutrien utilization degree, measure the green degree index of plant when last imaging before blooming.On the contrary, under drought stress growth conditions, the green degree index of plant while measuring imaging first after arid.

plant

Term " plant " comprises ancestors and offspring and the plant part of whole strain plant, plant as used in this article, comprise seed, branch, stem, leaf, root (comprising stem tuber), flower and tissue and organ, wherein every kind of mentioned object comprises goal gene/nucleic acid.Term " plant " also comprises vegetable cell, suspension culture, callus, embryo, meristem zone, gametophyte, sporophyte, pollen and sporule, and same every kind of object of mentioning comprises goal gene/nucleic acid.

The plant being used in particular in the inventive method comprises the whole plants that belong to vegitabilia (Viridiplantae) superfamily, and especially monocotyledons and dicotyledons comprises and be selected from following feeding or feed beans, ornamental plant, food crop, tree or shrub: maple species (Acer spp.), Actinidia species (Actinidia spp.), Abelmoschus species (Abelmoschus spp.), sisal hemp (Agave sisalana), Agropyron species (Agropyron spp.), the bent grass (Agrostis stolonifera) of crawling, allium species (Allium spp.), Amaranthus species (Amaranthus spp.), Europe beach grass (Ammophila arenaria), pineapple (Ananas comosus), Anona species (Annona spp.), celery (Apium graveolens), Arachis species (Arachis spp.), Artocarpus Forst species (Artocarpus spp.), officinalis (Asparagus officinalis), Avena species (Avena spp.) (for example oat (Avena sativa), wild avena sativa (Avena fatua), than praising oat (Avena byzantina), Avena fatua var.sativa, hybrid oat (Avena hybrida), carambola (Averrhoa carambola), Ce Sinobambusa species (Bambusa sp.), wax gourd (Benincasa hispida), Brazil's chestnut (Bertholletia excelsea), beet (Beta vulgaris), Btassica species (Brassica spp.) (for example colea (Brassica napus), overgrown with weeds blue or green species (Brassica rapa ssp.) [rape, rape (oilseed rape), turnip (turnip rape)]), Cadaba farinosa, tea (Camellia sinensis), Canna generalis Bailey (Canna indica), hemp (Cannabis sativa), Capsicum species (Capsicum spp.), Carex elata, papaya (Carica papaya), carissa macrocarpa (Carissa macrocarpa), hickory species (Carya spp.), safflower (Carthamus tinctorius), Castanea species (Castanea spp.), America kapok (Ceiba pentandra), hare's-lettuce (Cichorium endivia), Cinnamomum species (Cinnamomum spp.), watermelon (Citrullus lanatus), both citrus species (Citrus spp.), cocoanut species (Cocos spp.), Coffea species (Coffea spp.), taro (Colocasia esculenta), Africa Firmiana species (Cola spp.), Corchorus species (Corchorus sp.), coriander (Coriandrum sativum), Corylus species (Corylus spp.), hawthorn species (Crataegus spp.), Stigma Croci (Crocus sativus), Cucurbita species (Cucurbita spp.), Cucumis species (Cucumis spp.), cynara scolymus species (Cynara spp.), Radix Dauci Sativae (Daucus carota), acutifoliate podocarpium herb species (Desmodium spp.), longan (Dimocarpus longan), Wild yam species (Dioscorea spp.), Diospyros species (Diospyros spp.), Echinochloa species (Echinochloa spp.), oil palm belongs to (Elaeis) (for example oil palm (Elaeis guineensis), America oil palm Elaeis (oleifera)), Finger-millet (Eleusine coracana), Plumegrass species (Erianthus sp.), loquat (Eriobotrya japonica), eucalyptus belongs to (Eucalyptus sp.), red young fruit (Eugenia uniflora), Fagopyrum species (Fagopyrum spp.), Fagus species (Fagus spp.), alta fascue (Festuca arundinacea), Fructus Fici (Ficus carica), cumquat species (Fortunella spp.), Fragaria species (Fragaria spp.), ginkgo (Ginkgo biloba), Glycine species (Glycine spp.) (for example soybean, soybean (Soja hispida) or soybean (Soja max)), upland cotton (Gossypium hirstum), Helianthus (Helianthus spp.) (for example Sunflower Receptacle (Helianthus annuus)), long tube tawny daylily (Hemerocallis fulva), hibiscus species (Hibiscus spp.), Hordeum (Hordeum spp.) (for example barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), Juglans species (Juglans spp.), lettuce (Lactuca sativa), Lathyrus species (Lathyrus spp.), Lens culinaris (Lens culinaris), flax (Linum usitatissimum), lichee (Litchi chinensis), Lotus species (Lotus spp.), patola (Luffa acutangula), lupinus species (Lupinus spp.), Luzula sylvatica, tomato species (Lycopersicon spp.) (for example tomato (Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme)), sclerderm Macroptilium species (Macrotyloma spp.), Malus species (Malus spp.), recessed edge Malpighia coccigera (Malpighia emarginata), shea (Mammea americana), mango (Mangifera indica), cassava species (Manihot spp.), sapota (Manilkara zapota), clover (Medicago sativa), Melilotus species (Melilotus spp.), Mentha species (Mentha spp.), awns (Miscanthus sinensis), Momordica species (Momordica spp.), black mulberry (Morus nigra), Musa species (Musa spp.), Nicotiana species (Nicotiana spp.), Olea species (Olea spp.), Opuntia species (Opuntia spp.), bird foot Macroptilium species (Ornithopus spp.), Oryza (Oryza spp.) (for example rice, broad-leaved rice (Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), Purple Granadilla (Passiflora edulis), Selinum pastinaca (Pastinaca sativa), Pennisetum species (Pennisetum sp.), Persea species (Persea spp.), celery (Petroselinum crispum), Phalaris grass (Phalaris arundinacea), Phaseolus species (Phaseolus spp.), timothy grass (Phleum pratense), thorn certain herbaceous plants with big flowers species (Phoenix spp.), south reed (Phragmites australis), Physalis species (Physalis spp.), Pinus species (Pinus spp.), Pistacia vera (Pistacia vera), Pisum species (Pisum spp.), Poa L. species (Poa spp.), Populus species (Populus spp.), mesquite grass species (Prosopis spp.), Prunus species (Prunus spp.), Psidium species (Psidium spp.), pomegranate (Punica granatum), European pear (Pyrus communis), oak species (Quercus spp.), radish (Raphanus sativus), rheum rhabarbarum (Rheum rhabarbarum), currant species (Ribes spp.), castor-oil plant (Ricinus communis), rubus species (Rubus spp.), saccharum species (Saccharum spp.), Salix species (Salix sp.), Sambucus species (Sambucus spp.), rye (Secale cereale), flax species (Sesamum spp.), sinapsis alba species (Sinapis sp.), Solanum (Solanum spp.) (for example potato (Solanum tuberosum), red eggplant (Solanum integrifolium) or tomato (Solanum lycopersicum)), dichromatism chinese sorghum (Sorghum bicolor), spinach species (Spinacia spp.), Syzygium species (Syzygium spp.), Tagetes species (Tagetes spp.), tamarind (Tamarindus indica), cocoa tree (Theobroma cacao), Clover species (Trifolium spp.), Triticale sp., Triticosecale rimpaui, Triticum species (Triticum spp.) (for example common wheat (Triticum aestivum), durum wheat (Triticum durum), cylinder wheat (Triticum turgidum), Triticum hybernum, Macha wheat (Triticum macha) (Triticum macha), common wheat (Triticum sativum) or common wheat (Triticum vulgare)), little Flower of Chinese Globeflower (Tropaeolum minus), Flower of Chinese Globeflower (Tropaeolum majus), genus vaccinium species (Vaccinium spp.), tare species (Vicia spp.), Vigna species (Vigna spp.), sweet violet (Viola odorata), Vitis species (Vitis spp.), Zea mays, Zizania palustris, zizyphus species (Ziziphus spp.) etc.

detailed Description Of The Invention

Now be surprised to find the expression of nucleotide sequence in plant that improves coding AMT polypeptide described herein and produced the plant with respect to control plant with the Correlated Yield Characters of enhancing.According to an embodiment, the invention provides the method that strengthens Correlated Yield Characters in plant with respect to control plant, it comprises the expression of the nucleotide sequence that improves coding AMT polypeptide in plant.

The preferred method of expressing for increasing the nucleotide sequence of coding AMT polypeptide is the nucleotide sequence of introducing and expressing coding AMT polypeptide in plant.

Hereinafter any mentioning of " can be used for the protein of the inventive method " all referred to AMT polypeptide defined herein.Hereinafter " can be used for the nucleotide sequence of the inventive method " any mentioned the nucleotide sequence of this AMT polypeptide that all refers to encode.The nucleotide sequence of plant to be introduced (therefore can be used for implementing the inventive method) is that coding is existing by any nucleotide sequence of the polypeptide type of describing, below also referred to as " AMT nucleotide sequence " or " AMT gene ".

" AMT polypeptide " defined herein refers to comprise with conserved domain (CD) shown in SEQ ID NO:33 and has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or any polypeptide of the structural domain of higher (preference degree improves gradually) amino acid sequence identity.

As an alternative or supplement, " AMT polypeptide " defined herein refers to comprise following any polypeptide: (i) the ammonium transporter structural domain of InterPro accession number IPR0001905; (ii) at least 10 transbilayer helixs.

As an alternative or supplement, " AMT polypeptide " defined herein refer in the time being used for building AMT phylogenetic tree (as shown in Figure 2), illustrates with comprising in SEQ ID NO:2(Fig. 2 with circle) shown in the clade of AMT polypeptide rather than any peptide sequence of any other AMT clade cluster of peptide sequence.

As an alternative or supplement, " AMT polypeptide " defined herein refers to have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or any polypeptide of higher (preference degree improves gradually) amino acid sequence identity with AMT polypeptide shown in SEQ ID NO:2 or the given peptide sequence of any this paper Table A.

As an alternative or supplement, " AMT polypeptide " can remedy the yeast strain MLY131 (Hildebrand (2005) J Phycol41:105-113) that lacks all 3 kinds of unartificial yeast ammonium transporters.

Term " structural domain " and " motif " define in this paper " definition " chapters and sections.There is the special database for the identification of structural domain, such as SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA95,5857-5864; Letunic etc. (2002) Nucleic Acids Res30; 242-244); InterPro (Mulder etc.; (2003) Nucl.Acids.Res.31; 315-318); Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings2nd International Conference on Intelligent Systems for Molecular Biology.Altman R., Brutlag D., Karp P.; Lathrop R., Searls D. writes, 53-61 page; AAAI Press, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004) or Pfam (Bateman etc., Nucleic Acids Research30 (1): 276-280 (2002).One group of instrument for Computer Analysis protein sequence can obtain from ExPASY protein groups server (Swiss Institute of Bioinformatics (Gasteiger etc., ExPASy:the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res.31:3784-3788 (2003)).The analysis of the peptide sequence to SEQ ID NO:2 is shown in embodiment 4 herein.For example, the ammonium transporter structural domain that AMT polypeptide shown in SEQ ID NO:2 comprises InterPro accession number IPR0001905.Can also use routine techniques (as sequence alignment) to identify structural domain.The comparison of Table A polypeptide is herein shown in Fig. 3.This comparison can be used for identifying structural domain the most conservative between AMT polypeptide, for example conserved domain shown in SEQ ID NO:33 (CD) (being contained in SEQ ID NO:2).

Aligned sequences is take the method that compares as known in the art, and these methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.It is the highest and make the minimum overall comparison of room number (in complete sequence) that GAP utilizes the algorithm of Needleman and Wunsch ((1970) J Mol Biol48:443-453) to find two sequence chien shihs couplings numbers.BLAST algorithm (Altschul etc. (1990) J Mol Biol215:403-10) calculates per-cent sequence identity and carries out the statistical analysis of similarity between two sequences.Provide to the public at National Centre for Biotechnology Information (NCBI) for the software that carries out BLAST analysis.Can use ClustalW multiple sequence alignment algorithm (1.83 editions) and per-cent point system as given tacit consent to pairing comparison parameter easily to identify homologue.Also can use one of method providing in MatGAT software package (Campanella etc. (2003), BMC Bioinformatics.10:29.MatGAT:an application that generates similarity/identity matrices using protein or DNA sequences) to determine overall similarity and identity per-cent.One skilled in the art will recognize that, can carry out a small amount of manual editing to optimize the comparison between conservative property motif.In addition, can also replace full length sequence to identify homologue with specific structural domain.Sequence identity value can be used the said procedure of default parameters to measure on complete nucleotide sequence or peptide sequence or on selected structural domain or conservative property motif.Embodiment 3 has herein described the per-cent identity between AMT polypeptide shown in SEQ ID NO:2 and the listed AMT polypeptide of Table A in table B, and it can be low to moderate 44% amino acid sequence identity.If carry out identity calculating between the conserved domain of AMT polypeptide shown in conserved domain (CD) shown in SEQ ID NO:33 (being contained in SEQ ID NO:2 and SEQ ID NO:4) and Table A and Fig. 3, per-cent identity can improve.The table B1 that the results are shown in the application of these calculating.

The work of Prediction of Protein Subcellular Location is very important, and fully research.Understand being positioned with of protein and help illustrate its function.The scope of protein positioning experimental technique is from immunolocalization to using green to entangle photoprotein (GFP) or β-glucuronidase (GUS) labelled protein.These methods are very accurate, but need a large amount of work compared with computer approach.From sequence data, protein positioning is carried out to computer forecast and recently obtained remarkable progress.The ExPASy proteomics instrument that algorithm comprises that Swiss Institute for Bioinformatics provides, such as PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, the TMHMM etc. of obtaining well known to those skilled in the art.The application's embodiment 5 has described the prediction of the Subcellular Localization to AMT polypeptide shown in SEQ ID NO:2.

In addition the AMT polypeptide (at least its natural form) that, can be used for the inventive method can carry out transmembrane transport to ammonium conventionally.There is much mensuration to measure this picked-up activity, what comprise endogenous ammonium transporter defective yeast bacterial strain remedies mensuration (Ninneman etc. (1994) EMBO J 13:3464-3471), and the picked-up in yeast, Xenopus Oocytes (Ludewig etc. (2003) J Biol Chem278:45603-45610), vegetable cell, roots of plants (Yuan etc. (2007) Plant Phys143:732-744) and complete plant (Hoque etc. (2006) Functional Plant Biology 33:153-163) is measured.

The present invention is illustrated with the nucleotide sequence shown in SEQ ID NO:3 (being contained in SEQ ID NO:1) conversion of plant, and its coding AMT peptide sequence SEQ ID NO:4(is contained in SEQ ID NO:2).But enforcement of the present invention is not limited to these sequences; Method of the present invention can advantageously utilize any AMT peptide coding nucleotide sequence defined herein to implement.

The example of AMT peptide coding nucleotide sequence provides in the Table A of this paper embodiment 1.Such nucleotide sequence can be used for implementing method of the present invention.The peptide sequence providing in the Table A of embodiment 1 is the straight homologues of AMT polypeptide and the exemplary sequence of paralog thing shown in SEQ ID NO:2 or SEQ ID NO:4, and wherein term " straight homologues " and " paralog thing " are as defined herein.Can easily find more straight homologuess and paralog thing by carrying out so-called mutual BLAST retrieval.Conventionally, this for example comprises, with search sequence (, utilizing the listed arbitrary sequence of Table A of embodiment 1) and carries out the BLAST first of BLAST for any sequence library as the ncbi database can the public obtaining.In the time starting from nucleotide sequence, conventionally use BLASTN or TBLASTX (utilizing standard default value), and in the time starting from protein sequence, use BLASTP or TBLASTN (utilizing standard default value).BLAST result can optionally be filtered.Then use full length sequence in result or the unfiltered result of filtering to carry out reverse BLAST (quadratic B LAST) (in the situation that search sequence is SEQ ID NO:1 or SEQ ID NO:2, quadratic B LAST will for Phaeodactylum tricornutum sequence) for the search sequence biological sequence of originating.Then first with the result of quadratic B LAST.If first in BLAST high rank hit the same species being derived from from search sequence, then oppositely BLAST causes search sequence ideally among the highest hitting, and identifies paralog thing; If first in BLAST high rank hit the same species not being derived from from search sequence, and preferably in the time of reverse BLAST, cause search sequence to be hit for the highest, identify straight homologues.

Hitting of high rank is low the hitting of E value.E value is lower, score value more remarkable (or in other words, chancing on this probability hitting lower).The calculating of E value is well-known in the art.Except E value, comparative result is also marked by identity percentage ratio.Identity per-cent refers to that two compare the number of the identical Nucleotide (or amino acid) on length-specific between nucleic acid (or polypeptide) sequence.The in the situation that of extended familys, ClustalW be can use, succeeded by coming so that the cluster of additional related gene is visual in abutting connection with tree, and to straight homologues and paralog thing identified.

Nucleic acid variant also can be used for implementing method of the present invention.The example of this class nucleic acid variant comprises the homologue of arbitrary peptide sequence and the nucleotide sequence of derivative shown in the Table A of the embodiment 1 that encodes, and wherein " homologue " and " derivative " as defined herein.The nucleotide sequence that can be used for equally the inventive method is straight homologues or the homologue of paralog thing and the nucleotide sequence of derivative of arbitrary peptide sequence shown in the Table A of coding embodiment 1.Can be used for the unmodified protein matter that the homologue of the inventive method is derived from it with derivative and there is substantially the same biological activity and functionally active.

Other nucleic acid variants that can be used for implementing the inventive method comprise the part of AMT peptide coding nucleotide sequence, the nucleotide sequence with AMT peptide coding nucleic acid array hybridizing, the splice variant of AMT peptide coding nucleotide sequence, the allelic variant of AMT peptide coding nucleotide sequence, and the variant of the AMT peptide coding nucleotide sequence obtaining by gene shuffling.Term hybridization sequences, splice variant, allelic variant and gene shuffling are as described herein.

AMT peptide coding nucleotide sequence is without being total length nucleotide sequence, because the enforcement of the inventive method does not rely on the use of total length nucleotide sequence.According to the present invention, the method that strengthens Correlated Yield Characters in plant is provided, has been included in plant the part of introducing and expressing the nucleotide sequence of straight homologues, paralog thing or the homologue of arbitrary peptide sequence shown in the part of arbitrary nucleotide sequence shown in the Table A of embodiment 1 or the Table A of coding embodiment 1.

For example, can be by nucleotide sequence being carried out to " part " that one or more disappearances are prepared described nucleotide sequence." part " can be used with the form separating, or itself and other coding (or non-coding) sequence can be merged, so that for example, produces the protein that has combined some activity.In the time merging with other encoding sequences, the polypeptide producing after translation may be larger than what predict for this protein portion.

" part " that the can be used for the inventive method AMT polypeptide as herein defined of encoding, and there is substantially the same biological activity with the peptide sequence providing in embodiment 1 Table A.Preferably " part " is the part of arbitrary nucleotide sequence of providing in embodiment 1 Table A, or the straight homologues of arbitrary peptide sequence providing in coding embodiment 1 Table A or the part of the nucleotide sequence of paralog thing.According to the relative importance value order increasing progressively, preferably should " part " length be at least 400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,1250,1300,1350,1380 or more continuous nucleotide, described continuous nucleotide is the arbitrary nucleotide sequence providing in embodiment 1 Table A, or the straight homologues of arbitrary peptide sequence or the nucleotide sequence of paralog thing that in coding embodiment 1 Table A, provide.Preferably " part " is the part that encoded packets contains the nucleotide sequence of the peptide sequence of following structural domain, and conserved domain (CD) shown in described structural domain and SEQ ID NO:33 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity according to the relative importance value order increasing progressively.More preferably " part " is a part for the nucleotide sequence of SEQ ID NO:1.Most preferably " part " represented by SEQ ID NO:3.

The another kind of nucleotide sequence variant that can be used for the inventive method is under the stringent condition reducing, preferably under stringent condition, can with the nucleotide sequence of the nucleic acid sequence encoding of AMT polypeptide defined herein or " part " hybridization defined herein.

According to the present invention, the method that strengthens plant biomass correlated character is provided, be included in plant introduce and express can with the nucleotide sequence of arbitrary nucleic acid array hybridizing shown in embodiment 1 Table A, or be included in plant, introduce and express can with the nucleotide sequence of the nucleic acid array hybridizing of straight homologues, paralog thing or the homologue of arbitrary peptide sequence shown in coding embodiment 1 Table A.

The hybridization sequences that the can be used for the inventive method AMT polypeptide as herein defined of encoding, and there is substantially the same biological activity with peptide sequence shown in embodiment 1 Table A.Preferably hybridization sequences can be hybridized with arbitrary nucleic acid array hybridizing shown in embodiment 1 Table A or with the part of arbitrary aforementioned sequence, and wherein part as hereinbefore defined; Or wherein hybridization sequences can with the straight homologues of arbitrary peptide sequence or the nucleic acid array hybridizing of paralog thing shown in coding embodiment 1 Table A.Preferably hybridization sequences can with the nucleic acid array hybridizing of encoded packets containing the peptide sequence of following structural domain, conserved domain (CD) shown in described structural domain and SEQ ID NO:33 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity according to the relative importance value order increasing progressively.Most preferably hybridization sequences can be hybridized with nucleotide sequence shown in SEQ ID NO:1 or its part.

The another kind of nucleotide sequence variant that can be used for the inventive method is the coding splice variant of defined AMT polypeptide above, and wherein splice variant as defined herein.

According to the present invention, the method that strengthens Correlated Yield Characters is provided, has been included in the splice variant of introducing and expressing the splice variant of arbitrary nucleotide sequence shown in embodiment 1 Table A or the nucleotide sequence of straight homologues, paralog thing or the homologue of arbitrary peptide sequence shown in embodiment 1 Table A of encoding in plant.

Preferred splice variant is the splice variant of nucleotide sequence shown in SEQ ID NO:1, or the splice variant of the straight homologues of coding SEQ ID NO:2 or the nucleotide sequence of paralog thing.Preferably splice variant is the splice variant that encoded packets contains the nucleotide sequence of the peptide sequence of following structural domain, and conserved domain (CD) shown in described structural domain and SEQ ID NO:33 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity according to the relative importance value order increasing progressively.

The another kind of nucleotide sequence variant that can be used for implementing the inventive method is the allelic variant of defined AMT peptide coding nucleotide sequence above, and wherein allelic variant as defined herein.

According to the present invention, the method that strengthens Correlated Yield Characters is provided, be included in the allelic variant of introducing and expressing arbitrary nucleotide sequence shown in embodiment 1 Table A in plant, or be included in the allelic variant of introducing and expressing the nucleotide sequence of straight homologues, paralog thing or the homologue of arbitrary peptide sequence shown in coding embodiment 1 Table A in plant.

Can be used for the allelic variant of the inventive method and the AMT polypeptide of SEQ ID NO:2 and there is substantially the same biological activity with any peptide sequence described in embodiment 1 Table A.The natural existence of allelic variant, and in method of the present invention, comprise these natural allelotrope of use.Preferably allelic variant is the allelic variant of SEQ ID NO:1, or the allelic variant of the straight homologues of coding SEQ ID NO:2 or the nucleotide sequence of paralog thing.Preferably allelic variant is the allelic variant of the peptide sequence that comprises following structural domain, and conserved domain (CD) shown in described structural domain and SEQ ID NO:33 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity according to the relative importance value order increasing progressively.

Gene shuffling or orthogenesis also can be used for producing the variant of defined AMT peptide coding nucleotide sequence above; Wherein term " gene shuffling " as defined herein.

According to the present invention, the method that strengthens Correlated Yield Characters is provided, be included in the variant of introducing and expressing arbitrary nucleotide sequence shown in embodiment 1 Table A in plant, or be included in the variant of introducing and expressing the nucleotide sequence of straight homologues, paralog thing or the homologue of the arbitrary peptide sequence providing in coding embodiment 1 Table A in plant, wherein said variant nucleic acid sequences obtains by gene shuffling.

Preferably, the variant nucleic acid sequences encoded packets obtaining by gene shuffling is containing the peptide sequence of following structural domain, and conserved domain (CD) shown in described structural domain and SEQ ID NO:33 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity according to the relative importance value order increasing progressively.

In addition, also can utilize site-directed mutagenesis to obtain nucleotide sequence variant.Some methods can be used to realize site-directed mutagenesis, and modal is the method (Current Protocols in Molecular Biology, Wiley edits) of PCR-based.

The nucleotide sequence of coding AMT polypeptide can be from any natural or artificial source.Nucleic acid can be modified its natural form in composition and/or genome environment by the manual operation of having a mind to.The nucleotide sequence of coding AMT polypeptide is from eukaryote field, preferably from vesica algae circle (Chromalveolata kingdom), more preferably from not waiting flagellum door (Heterokontophyta phylum).More preferably encode the nucleotide sequence of AMT polypeptide from Diatomacae (Bacillariophyceae(diatoms) class), for example, from following order: bent shell Cutleriales (Achnanthales), diatom order (Bacillariales), centrales (Centrales) (for example Thalassiosira pseudonana (Thalassiosira pseudonana)), the curved Cutleriales of bridge (Cymbellales), short seam Cutleriales (Eunotiales), septum pectorale Cutleriales (Mastogloiales), boat-shaped Cutleriales (Naviculales), Pennales (Pennales) (for example Phaeodactylum tricornutum (Pheaodactylum tricornutum)), rod bar Cutleriales (Rhopalodiales), two water chestnut Cutleriales (Surirellales) or Thalassiophysales.Most preferably encode the nucleotide sequence of AMT polypeptide from Phaeodactylum tricornutum.

The enforcement of the inventive method produces the plant with respect to control plant with the Correlated Yield Characters of enhancing.In " definition " part in this article, term " output " and " seed production " are described in more detail.

Take corn as example, output increase can show as following one or more indexs: the increase of the increase, the increase of every strain plant spike number, the increase of line number, every row grain number of built vertical plant number in per hectare or acre, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that full seed number is total and be multiplied by 100 divided by seed) and other.Take rice as example, output increase itself can show as the increase of following one or more indexs: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (little Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be full seed number divided by seed sum and be multiplied by 100), the increase of thousand seed weight and other.

The invention provides the method that strengthens the Correlated Yield Characters of plant with respect to control plant, described method comprises the expression of the nucleic acid sequence encoding that improves AMT polypeptide defined herein in plant.

Because transgenic plant of the present invention have the Correlated Yield Characters of enhancing, thereby on corresponding stage in its life cycle with respect to the growth velocity of control plant, these plants likely show increase growth velocity (its life cycle at least partly during).

The growth velocity increasing can be specific for one or more parts (comprising seed) of plant, or can substantially spread all over whole strain plant.The plant with the growth velocity of increase can possess shorter life cycle.The life cycle of plant can be considered as meaning the needed time in stage that grows to plant and produced the dry mature seed similar to parent material from dry mature seed.This life cycle can be affected by following factors, as early stage vigor, growth velocity, green degree index, flowering time and seed maturity speed.The increase of growth velocity can on the one or more stages in plant life cycle or occur during whole plant life cycle substantially.The growth velocity increasing during early stage in plant life cycle can reflect (in early days) vigor of raising.The increase of growth velocity can change the harvest cycle of plant, allow plant compared with late sowing kind and/or compared with early harvest, otherwise this is by impossible (similar effect can obtain with flowering time early, and the flowering time of delay is not the proterties of wanting in crop conventionally).If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow subsequently and gather in the crops other rice plant, all rice plant is all within a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing harvesting corn plant, for example sowing subsequently optional results soybean, potato or any other suitable plant) of different plant species.It is also possible from identical rhizome, gathering in the crops additional times in the situation of some crop plants.The harvest cycle that changes plant can cause the increase of year biomass yield of every acre (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow than its wild type counterparts cultivating transgenic plant in geographic area widely, because the region limits of cultivating crop is often determined by the plantation time (early season) or in the adverse environment condition of results period (season in evening).If shortening harvest cycle, can avoid this class unfavourable condition.Growth velocity can be determined by obtain many kinds of parameters from growth curve, this type of parameter can be: T-Mid (plant reaches the time that its 50% overall dimension spends) and T-90 (plant reaches the time that its 90% overall dimension spends), etc.

According to preferred feature of the present invention, the enforcement of the inventive method produces the plant with respect to control plant with the growth velocity of increase.Thereby according to the present invention, providing the method that increases plant growth rate, described method comprises increases the expression of the nucleotide sequence of coding AMT polypeptide as defined herein in plant.

Compared with the control plant of growing under comparable conditions, no matter under non-stress condition or plant is exposed under various abiotic stress, all there is the Correlated Yield Characters of enhancing in plant.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under condition of serious stress of soil condition, plant even can stop growing completely.On the other hand, slightly coerce and be defined as in this article plant any of its exposure coerced, wherein said coercing do not cause plant to stop growing completely and do not recover the ability of growth.Compared with control plant under non-stress condition, slightly coerce and in meaning of the present invention, cause being coerced plant-growth and reduce and be less than 40%, 35% or 30%, be preferably less than 25%, 20% or 15%, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower.Due to the progress on agricultural practice (irrigation, fertilising, pesticide treatments), in raise crop plant, often do not run into condition of serious stress of soil.Therefore, by the impaired growth of the slight stress-inducing upper undesirable feature of agricultural often.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Abiotic stress can be because of due to arid or waterlogging, Anoxia stress, salt stress, chemical toxicity, oxidative stress and heat, cold or freezing temperature.Abiotic stress can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress causing.It is generally that those that caused as bacterium, virus, fungi, nematode and insect by pathogenic agent are coerced that biology is coerced.Term " non-coercing " condition is those envrionment conditionss that allow plant optimum growh as used in this article.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method, with respect to the control plant of cultivating under comparable conditions, is given the Correlated Yield Characters of the plant enhancing of cultivating under non-stress condition or under gentle stress conditions.Thereby according to the present invention, in the plant that is provided for cultivating under non-stress condition or under gentle stress conditions, strengthening the method for Correlated Yield Characters, described method comprises the expression of the nucleotide sequence that improves coding AMT polypeptide in plant.

The enforcement of the inventive method, with respect to the control plant of cultivating under comparable stress conditions, is given the Correlated Yield Characters of the plant enhancing of cultivating under abiotic stress condition.As report in (Planta (2003) 218:1-14) such as Wang, abiotic stress causes adversely affecting a series of morphological change, physiology variation, biochemical change and the molecule of plant-growth and productivity to change.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described drought stress and high salinity and have coerced " intersection " of a very high degree.For example, arid and/or salinification main manifestations are osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar Cell signal transduction pathway and cell response, as produced stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Because different environment-stress activates similar approach, therefore the example that the present invention carries out with regard to drought stress should not be regarded as and be confined to drought stress, shows that AMT polypeptide defined above participates in the demonstration that improves Correlated Yield Characters with respect to the control plant of cultivating under suitable stress conditions under general abiotic stress but more should regard as.

Term defined herein " abiotic stress " refers to following any or multiple: water coerces that (due to arid or water excess), anoxic are coerced, salt stress, temperature stress (due to hot, cold or freezing temperature), chemical toxicity are coerced and oxidative stress.According to an aspect of the present invention, abiotic stress is osmotic stress, and it is selected from, and water is coerced, salt stress, oxidative stress and ion are coerced.Preferably, water is coerced as drought stress.Term " salt stress " is not limited only to common salt (NaCl), but following any the coercing that one or more cause of can serving as reasons: NaCl, KCl, LiCl, MgCl ₂, CaCl ₂deng.

The Correlated Yield Characters of plant raising for the control plant of cultivating under suitable condition of cultivating under abiotic stress condition is given in the enforcement of the inventive method.Therefore, according to the present invention, provide the method that improves Correlated Yield Characters under abiotic stress condition in the plant of cultivating, the method is included in the expression that improves the nucleotide sequence of coding AMT polypeptide in plant.According to an aspect of the present invention, abiotic stress is osmotic stress, one or more that it is selected from, and water is coerced, salt stress, oxidative stress and ion are coerced.

Another example that abiotic environment is coerced is the reduction of one or more required nutrien utilization degree of the assimilation of plant-growth and growth.Because nutrientuse efficiency has remarkably influenced to plant biomass and quality product, therefore use a large amount of fertilizer to optimize plant-growth and quality in field.The productivity of plant is generally limited to three kinds of Main Nutrients: phosphorus, potassium and nitrogen, in this three, nitrogen is the speed limit element of plant-growth normally.Therefore, the required Main Nutrients element of plant-growth is nitrogen (N).This is the component that is found in the multiple important compound in viable cell, and described compound comprises amino acid, protein (enzyme), nucleic acid and chlorophyll.Approximately 16% of 1.5% to 2% and total plant protein of plant dry matter is nitrogen.Therefore, the availability of nitrogen is the key constraints (Frink etc. (1999) Proc Natl Acad Sci USA96 (4): 1175-1180) of crop plants growth and production, and also protein accumulation and amino acid composition is had to great effect.The crop plants while therefore, cultivation under the limited condition of nitrogen with the Correlated Yield Characters of raising is highly significant.

The Correlated Yield Characters of raising is given under nutrien utilization degree reduction condition plant that (particularly under the condition of the nitrogen availability reducing) cultivate and is had for the control plant of cultivating under suitable condition in the enforcement of the inventive method.Therefore, according to the present invention, the method that improves Correlated Yield Characters in the plant that (preferably under the condition of the nitrogen availability reducing) cultivates under nutrien utilization degree reduction condition is provided, and the method is included in the expression that improves the nucleotide sequence of coding AMT polypeptide in plant.Reduce nutrien utilization degree may be due to nutrient (as nitrogen, phosphorus and other P contained compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron etc.) lack or surplus due to.Preferably, the nutrien utilization degree of reduction is the nitrogen availability reducing.

The present invention includes plant or its part (comprising seed) or its cell that can be obtained by the method according to this invention.The nucleic acid transgenosis that described plant or its part or its cell contain coding AMT polypeptide as hereinbefore defined.

The present invention also provides genetic constructs and carrier, is beneficial in plant, introduce the nucleic acid sequence encoding of AMT polypeptide as hereinbefore defined and/or improve its expression.Gene construct insertion can be suitable for to conversion and enter plant the carrier for the cells goal gene in conversion, this carrier can be commercially available carrier.The present invention also provides gene construct purposes in the methods of the invention as herein defined.

More specifically, the invention provides such construct, it contains:

(a) nucleotide sequence of coding AMT polypeptide as hereinbefore defined;

(b) one or more control sequences that can improve the expression of (a) amplifying nucleic acid sequence; With optional

(c) transcription termination sequence.

The nucleotide sequence of optimized encoding AMT polypeptide is as above definition.Term " control sequence " and " terminator sequence " are as defined herein.

Preferably one of control sequence of construct is the constitutive promoter separating from Plant Genome.An example of plant constitutive promoter is GOS2 promotor, is preferably rice GOS2 promotor, more preferably GOS2 promotor shown in SEQ ID NO:34.

The carrier conversion of plant that use contains any above-mentioned nucleotide sequence.Technician fully knows the genetic elements that must exist in carrier, to successfully transform, select and breed the host cell containing aim sequence.Aim sequence is effectively connected in to one or more control sequences (being at least connected in promotor).

Advantageously, no matter the promotor of any type, be natural or synthetic, the expression that all can be used for improving nucleotide sequence.Constitutive promoter, preferably from Plant Genome separate constitutive promoter be particularly useful for method of the present invention.Plant constitutive promoter drives encoding sequence with following horizontal expression, and described level is lower than the level obtaining under the control at 35S CaMV viral promotors in all cases.

Other organ specific promoters, for example in leaf, stem, stem tuber, meristematic tissue, seed (embryo and/or endosperm), the preferential promotor of expressing can be used for implementing method of the present invention." definition " part is herein shown in the definition of multiple promotor type.

Should be clear and definite, suitability of the present invention is not limited to the nucleotide sequence of the coding AMT polypeptide as shown in SEQ ID NO:1 or SEQ ID N:3, the expression of AMT peptide coding nucleotide sequence when suitability of the present invention is also not limited to be driven by constitutive promoter.

Optionally, can in the construct of introduced plant, use one or more terminator sequences.Other regulatory elements can comprise transcribes and translational enhancer.Those skilled in the art will appreciate that and be suitable for implementing terminator of the present invention and enhancer sequence.Also can, described in definitional part, intron sequences be added in 5 ' non-translational region (UTR) or encoding sequence, to be increased in the amount of the ripe information of accumulating in cytosol.Other control sequence (except promotor, enhanser, silencer, intron sequences, 3 ' UTR and/or 5 ' UTR district) can be protein and/or RNA stable element.One skilled in the art will recognize that or can easily obtain this type of sequence.

Genetic constructs of the present invention can also comprise need to be used for the replication origin sequence that maintains and/or copy in particular cell types.An example is for example, in the time that needs are maintained genetic constructs additive type genetic elements (plasmid or clay molecule) in bacterial cell.Preferred replication origin includes but not limited to f1-ori and colE1.

For detecting as the successful transfer of nucleotide sequence used in the methods of the invention and/or the transgenic plant that selection comprises these nucleotide sequences, applying marking gene (or reporter gene) is favourable.Thereby genetic constructs can optionally comprise selectable marker gene.Selective marker has more detailed description in this paper " definition " part.

Known to nucleotide sequence is stable or integration,temporal during to vegetable cell, the only cellular uptake foreign DNA of small portion and be integrated into as required cellular genome, this depends on the rotaring dyeing technology of expression carrier used thereof and use.For identifying and select these intasomies, conventionally the gene of codes selection mark (one of as described above) is introduced to host cell together with goal gene.These marks can be for example therein these genes because using in the non-functional mutant of disappearance due to ordinary method for example.In addition, the nucleotide sequence molecule of codes selection mark can be introduced in host cell, with the sequence of polypeptide used in code book invention polypeptide or the inventive method in identical carrier, or on independent carrier.Can be for example identify (for example thering is the cell survival of selective marker of integration and other necrocytosis) by selection with the cell of the nucleotide sequence stable transfection of introducing.Once no longer need, can remove or excise marker gene from transgenic cell.The technology of removing for marker gene is known in the art, and useful technology is described in definitional part above.

The present invention also provides to produce with respect to control plant has the methods of the transgenic plant of the Correlated Yield Characters of enhancing, and it is included in any nucleotide sequence of introducing in plant and expressing coding and define AMT polypeptide above.

More specifically, the invention provides and produce the method with respect to control plant with the transgenic plant of the Correlated Yield Characters of enhancing, described method comprises:

(i) to the nucleotide sequence of introducing and expressing the coding AMT polypeptide under the control of plant constitutive promoter in plant, plant part or vegetable cell; With

(ii) culturing plants cell, plant part or plant under the condition of Promoting plant growth and growth.

(i) nucleotide sequence can be any nucleotide sequence of AMT polypeptide defined above of can encoding.

Can be by direct nucleotide sequence introduced plant cell or plant itself (comprising tissue, organ or any other parts of introduced plant).The preferred aspect according to the present invention, preferably by transforming nucleotide sequence introduced plant.Term " conversion " has more detailed description in this paper " definition " part.

All method regeneration that the vegetable cell of genetic modification can be familiar with by technician.Suitable method be found in above-mentioned S.D.Kung and R.Wu, Potrykus or

publication with Willmitzer.

Conventionally after transforming, select the vegetable cell or the cell mass that there are one or more marks, described mark is encoded by the expressive gene of plant moving with goal gene corotation, then the material regeneration of conversion is become to whole plant.For the plant of selecting to transform, conventionally the vegetable material obtaining in conversion process is placed under selective conditions, thereby the plant of conversion and non-transformed floral region can be separated.For example, can plant the seed obtaining in the above described manner, and after initial vegetative period, by spraying, it be carried out to suitable selection.Another may scheme be to use suitable selective agent, seed (suitably time after sterilizing) is planted on agar plate, thereby the seed only transforming can grow up to plant.Alternatively, existence that can selective marker (mark as described above) for the foliage filter screening transforming.

After DNA transfer and regeneration, also can evaluate the plant of inferring conversion, for example, analyze (southern blotting technique) with Southern, evaluate existence, copy number and/or the genome structure of goal gene.Alternatively or extraly, available Northern and/or Western analyze the expression level of the new DNA introducing of (western blotting) monitoring, and these two kinds of technology are all well known to those of ordinary skill in the art.

The conversion of plant producing can be bred in several ways, as passed through clonal propagation or classical breeding technique.For example, the plant that the first-generation (or T1) transforms can selfing, select the s-generation (or T2) transformant of isozygotying, and T2 plant can further breed by classical breeding technique.The inverting biological body producing can have various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's transformant (for example all cells contains expression cassette through transforming); The graft (for example, in plant, the root stock grafting of conversion is to non-transformed scion) of conversion and non-transformed tissue.

Any vegetable cell or plant that the present invention obviously prolongs and produced by any method described herein, and all plant parts and propagulum thereof.The present invention also prolongs and the offspring of cell, tissue, organ or the whole plant of the primary conversion that produced by any aforesaid method or transfection, and unique requirement is that described offspring presents genotype and/or the phenotypic characteristic identical with the parent who produces in the methods of the invention.

The present invention also comprises that described nucleotide sequence is effectively connected with plant constitutive promoter containing the separative coding host cell of the nucleotide sequence of defined AMT polypeptide above.According to the present invention, preferred host cell is vegetable cell.For the host plant of the nucleotide sequence for the inventive method or carrier, expression cassette or construct or carrier, it is in principle advantageously for synthesizing all plants of the polypeptide using in the methods of the invention.

The inventive method is advantageously applicable to any plant.The plant being used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprises feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, rape, clover, rape, cotton, tomato, potato and tobacco.Also preferably, plant is monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

The present invention also extends to the AMT(that comprises the separation being effectively connected with plant constitutive promoter as definition above) the gathered in the crops part of the plant of nucleic acid sequence encoding as, but be not limited to seed, leaf, fruit, flower, stem, rhizome, stem tuber and bulb.The invention further relates to derived from, preferably directly derived from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

All be recorded in this area for the method that improves nucleotide sequence or gene or gene product expression, its example provides in definitional part.

As described above, be by introduce and express AMT peptide coding nucleotide sequence plant for improving the preferred method of one of AMT peptide coding nucleotide sequence expression; But, also can realize the effect (strengthening Correlated Yield Characters) of implementing described method by other known technology, described technology includes but not limited to that T-DNA activates label, TILLING, homologous recombination.Being described in definitional part of these technology provides.

The present invention also comprises that AMT peptide coding nucleotide sequence as described herein and these AMT polypeptide are under normal growth condition, under abiotic stress growth (preferably osmotic stress growth conditions) condition, under the growth conditions of nutrien utilization degree reducing, preferably, under the condition of the nitrogen availability reducing, improve the purposes of any aforementioned Correlated Yield Characters in plant.

Can in the procedure of breeding, use nucleotide sequence or the AMT polypeptide itself of coding AMT polypeptide described herein, the DNA marker that wherein evaluation may be chain with AMT peptide coding gene genetic.Can use described gene/nucleotide sequence or described AMT polypeptide definition molecule marker itself.Then this DNA or protein labeling can be used in the procedure of breeding, to select in the method for the invention to have the plant of Correlated Yield Characters of enhancing as hereinbefore defined.

Gene/nucleotide sequence allelic variant of coding AMT polypeptide also can be in the auxiliary procedure of breeding of mark.This procedure of breeding needs to introduce allelic variation by using for example EMS mutagenesis to make mutagenic treatment to plant sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called causing " nature " origin.Carry out subsequently the evaluation of allelic variant, for example, by PCR method.After this be the step of discussing and cause the excellent allelic variant of the sequence of the Correlated Yield Characters strengthening for selecting.The growth performance that generally contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented selection.Can be in greenhouse or monitor on field growth performance.Other optional step comprises and will identify plant and the another kind of plant hybridization of excellent allelic variant.This can be used for for example producing the combination of target phenotypic characteristic.

The nucleotide sequence of coding AMT polypeptide also can be as probe to carry out genetic mapping and physical mapping to gene, and described probe is as the mark of a part for described gene and the proterties associated with these genes.This type of information can be in plant breeding, so that exploitation has the strain of wanting phenotype.This purposes of the nucleotide sequence of coding AMT polypeptide only needs to have the nucleotide sequence of at least 15 length of nucleotides.The nucleotide sequence of coding AMT polypeptide can be used as restriction fragment length polymorphism (RFLP) mark.Southern trace (the Sambrook J of the plant genome DNA of restrictive diges-tion, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) can survey with the nucleotide sequence of coding AMT polypeptide.Produce combination graphic can use subsequently computer program as MapMaker (Lander etc. (1987) Genomics1:174-181) carry out genetic analysis with build genetic map.In addition, this nucleotide sequence can be used for surveying the Southern trace containing through the genomic dna of one group of individuality of restriction endonuclease processing, and one group of wherein said individuality represents parental generation and has the offspring of definite genetic cross.The separation of DNA polymorphism is marked and is used for the nucleotide sequence of calculation code AMT polypeptide in the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) using in this colony previous genetic map obtaining.

Generation and its purposes in genetic mapping of the derivative probe of plant gene have been described in Bernatzky and Tanksley (1986) Plant Mol.Biol.Reporter4:37-41.Numerous publications have been described and have been used methodology mentioned above or the genetic mapping of its modification method to specific cDNA clone.For example, to hand over mutually group, the group that backcrosses, panmictic population, contiguous isozygotying be can be for mapping with other population of individuals to F2.This type of methodology is well known to the skilled person.

Described nucleotide sequence probe can (be also the arrangement of sequence on physical map for physical mapping; See that Hoheisel etc. exists: Non-mammalian Genomic Analyasis:A Practical Guide, Academic press1996,319-346 page and the reference of wherein quoting).

In another embodiment, nucleotide sequence probe can use directly entangling in light in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).Although current FISH graphing method preference is used large-scale clone, (several kb are to a hundreds of kb; See (1995) the Genome Res.5:13-20 such as Laan), but the improvement of sensitivity can allow to use shorter probe to carry out FISH mapping.

The method that is used for the multiple nucleic acid sequence based amplification of genetic mapping and physical mapping can be used described nucleotide sequence and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med11:95-96), pcr amplified fragment, Sheffield etc. (1993) Genomics16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic acid sequence Res.18:3671), Radiation hybrid mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic acid sequence Res.17:6795-6807).For these methods, the primer pair that designs and be created in amplified reaction or use in primer extension reaction by a kind of sequence of nucleotide sequence.The design of this type of primer is well known to the skilled person.Using in the method for PCR-based genetic mapping, may identify the DNA sequence dna difference that mapping intersects between parental generation in the region corresponding to current nucleotide sequence.But,

This is conventionally optional for graphing method.

The inventive method produces has the plant that Correlated Yield Characters improves as previously described.These proterties also can with other economic favourable proterties combination, as other output strengthen proterties, to abiotic stress and biological patience of coercing, patience to weedicide, sterilant, regulate the proterties of multiple constructivity feature and/or biochemical characteristics and/or physiologic character.

Accompanying drawing explanation

With reference to the following drawings, the present invention is described, in the accompanying drawings:

Fig. 1 representative is for the figure output of the TMHMM2.0 algorithm of SEQ ID NO:2.According to the prediction of algorithm, the N end of this polypeptide is positioned at film outside (cytosol is outer), is then 11 transbilayer helixs, and the C end of this polypeptide is positioned at the inner side (cytosol) of film.

Fig. 2 shows the phylogenetic tree from the biological AMT polypeptide in multiple source.Group I representative can be used for implementing the peptide sequence cluster of the inventive method, illustrates with bracket.Circle representative can be used for implementing the tapping point in tree between the polypeptide of the inventive method and other AMT polypeptide.

Fig. 3 shows AlignX (from Vector NTI10.3, the Invitrogen Corporation) Multiple Sequence Alignment to AMT polypeptide in Table A.Starting point and the terminal of conserved domain (CD) show with bracket, as shown in SEQ ID NO:33, and mark with X below consensus sequence.Frame has gone out the conservative property G(Gly that participates in normal AMT function) residue (Ludewig etc. (2003) J Biol Chem278:45603-10).

Fig. 4 shows the binary vector for improve the expression of the nucleotide sequence of coding AMT polypeptide under controlling in rice GOS2 promotor (pGOS2) rice (Oryza sativa).

Fig. 5 detailed example can be used for implementing the sequence of the inventive method.

Embodiment

The present invention describes with reference to following examples, and described embodiment is only for setting forth.Following examples are not intended to definition comprehensively or otherwise limit the scope of the invention.

DNA operation: except as otherwise noted, according to (Sambrook (2001) Molecular Cloning:a laboratory manual, the third edition, Cold Spring Harbor Laboratory Press, CSH, New York) or Ausubel etc. (1994), Current Protocols in Molecular Biology, described in Current Protocols the 1st volume and the 2nd volume, standard scheme carries out recombinant DNA technology.Be described in the Plant Molecular Biology Labfax (1993) being write by R.D.D of BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK) publication for the standard material of plant molecular work and method.

embodiment 1: identify the sequence relevant to the nucleotide sequence using in the inventive method

Usage data storehouse sequence retrieval instrument, as basic Local Alignment instrument (BLAST) (Altschul etc. (1990) J.Mol.Biol.215:403-410; With (1997) Nucleic Acids Res.25:3389-3402 such as Altschul) identify the sequence relevant to the nucleotide sequence using in the inventive method (full-length cDNA, EST or genome sequence) in those sequences of safeguarding in the Entrez RiboaptDB of NCBI (NCBI).This program be used for by nucleotide sequence or peptide sequence and sequence library relatively and the significance,statistical mating by calculating find the region between sequence with local similarity.For example, the polypeptide coded to nucleotide sequence of the present invention uses TBLASTN algorithm, adopts default setting and filter to ignore low-complexity sequence to start (set off).The result of analyzing relatively shows by pairing property, and according to probability score (E-value) sequence, wherein this scoring reflects the probability (E-value lower, the significance of hitting higher) of specific comparison result because accidentally occurring.Except E-value, more also score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number within the scope of length-specific between nucleotide sequence (or polypeptide) sequence.In some cases, can adjust default parameters to regulate the severity of retrieval.For example, can improve E-value and show strict coupling still less.Can identify by this short accurate coupling that approaches.

Table A provides the list of the nucleotide sequence relevant to the nucleotide sequence using in the inventive method.

Table A: the example of AMT peptide sequence and nucleic acid sequence encoding:

In some cases, relevant sequence is temporarily combined by for example genome research association (TIGR) of research association and to public.Can use eukaryotic gene straight homologues (EGO) database to identify this class correlated series, carry out keyword search or BLAST algorithm with interested nucleotide sequence or peptide sequence.In other cases, for concrete biopoiesis specific GenBank, for example for example create for Thalassiosira pseudonana and Phaeodactylum tricornutum by engaging genome association (Joint Genome Institute).

the comparison of embodiment 2:AMT peptide sequence

Use AlignX algorithm (from Vector NTI10.3, Invitrogen Corporation) to carry out the Multiple Sequence Alignment of all AMT peptide sequences in Table A.Comparison result is showed in Fig. 3 of the application.Use bracket to show for example beginning and the end of the conserved domain (CD) as shown in SEQ ID NO:33, and with X mark under consensus sequence.Mark the conservative G(Gly that relates to correct AMT function with square frame) residue (Ludewig etc. (2003) J Biol Chem278:45603-10).

embodiment 3: calculate the overall identity hundred between the peptide sequence for implementing the inventive method proportion by subtraction

Use one of the obtainable method in this area MatGAT (matrix overall comparison instrument) software (BMC Bioinformatics.20034:29.MatGAT:an application that generates similarity/identity matrices using protein or DNA sequences.Campanella JJ, Bitincka L, Smalley J.; Software is provided by Ledion Bitincka) be identified for implementing overall similarity percentage ratio and identity percentage ratio between the full-length polypeptide sequence of the inventive method.MatGAT software is that DNA sequence dna or protein sequence produce similarity/identity matrix, without the pre-comparison of data.It is a series of by comparison that this program is used Myers and Miller overall comparison algorithm (point penalty 12 is opened in room and point penalty 2 is extended in room) to carry out, and uses for example Blosum62 (for polypeptide) calculating similarity and identity and subsequently result be placed in to distance matrix.Sequence similarity shows in marginal lower part, and sequence identity shows in the marginal upper part in diagonal angle.

Parameter used is relatively:

Rating matrix: Blosum62

The first room: 12

Extend room: 2

Overall similarity in peptide sequence (exclusive segment peptide sequence) total length and identity software analysis result show in table B.

Between the conserved domain (CD) of (and being contained in SEQ ID NO:2 and SEQ ID NO:4) shown in SEQ ID NO:33 and the conserved domain (highlighting in as Fig. 3) of Table A polypeptide, carry out identical analysis, the results are shown in table B1.

Can be used for implementing identity per-cent between the full-length polypeptide sequence of the inventive method and can be low to moderate compared with SEQ ID NO:2 44% amino acid identity.

As show as shown in B1, the identity per-cent between the conserved domain (CD) of (and being contained in SEQ ID NO:2 and SEQ ID NO:4) shown in SEQ ID NO:33 and the conserved domain (as highlighted in Fig. 3) of Table A polypeptide is increased to 55% amino acid identity.

Embodiment 4: identify and can be used for implementing the structural domain that comprises in the peptide sequence of the inventive method

The integrated resource in protein families, structural domain and site (InterPro) database is the integrated interface of the common tag database used of the retrieval based on text and sequence.InterPro database has combined these databases, and described database uses different methods to learn with the biological information of relevant fully profiling protein matter in various degree to obtain protein tag.Cooperation database comprises SWISS-PROT, PROSITE, TrEMBL, PRINTS, Panther, ProDom and Pfam, Smart and TIGRFAMs.Interpro is provided by the European bioinformation institute of Britain.

InterPro scanning is if the result of the peptide sequence of SEQ ID NO:2 representative is in table C.

The InterPro scanning result of peptide sequence shown in table C:SEQ ID NO:2

Embodiment 5: can be used for the Subcellular Localization prediction of the peptide sequence of implementing the inventive method

The scope of protein positioning experimental technique is from immunolocalization to using green to entangle photoprotein (GFP) or β-glucuronidase (GUS) labelled protein.For example, use GFP fusion experiment that the AMT translocator from Arabidopis thaliana is positioned to (Yuan etc. (2003) Plant Cell 19:2636-2652) in plasma membrane.

Also carry out from sequence data the computer forecast to protein positioning.The ExPASy protein science instrument providing from bioinformation institute of Switzerland (Swiss Institute for Bioinformatics) can be provided algorithm well known to those skilled in the art, for example PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM and other.

The single cross-film α spiral of membrane spaning domain ordinary representation transmembrane protein.Because can being independent of the remainder of protein, the alpha-helix in film folds, so it is known as " structural domain ".More broadly, membrane spaning domain is thermodynamically stable any three dimensional protein structure in film.This can be the single α spiral of GA, stable compound, cross-film β bucket, β-spiral or any other structure of several cross-films α spiral.

Approximately 20 amino acid of transbilayer helix normal length, although they can much longer or much shorter.TMHMM2.0 be a kind of can predicted protein matter in the algorithm of transbilayer helix.The trustship on the server of Technical University Of Denmark (Technical University of Denmark) of this algorithm.Following table D has shown the TMHMM2.0 Output rusults that uses the peptide sequence information of SEQ ID NO:2 to obtain.According to prediction, the N-end of polypeptide is positioned at the outside (cytosol is outer) of film, is 11 transbilayer helixs afterwards, and the peptide C-end of polypeptide is arranged in the inner side (cytosol) of film.Identical configuration is applicable to SEQ ID NO:4, and difference is that the first outside is less.Fig. 1 is the diagram of Output rusults in table D.

Table D: the TMHMM2.0 Output rusults that uses the peptide sequence information of SEQ ID NO:2 to obtain.

Location	Amino acid coordinate
		Outside	1-53
TM spiral (TMhelix)	54-76
		Inner side	77-87
TMhelix	88-110
		Outside	111-131
TM spiral	132-154
		Inner side	155-160
TM spiral	161-183
		Outside	184-202
TM spiral	203-225
		Inner side	226-245
TM spiral	246-268
		Outside	269-282
TM spiral	283-305
		Inner side	306-317
TM spiral	318-340
		Outside	341-344
TM spiral	345-367
		Inner side	368-379
TM spiral	380-402
		Outside	403-429
TM spiral	430-452
		Inner side	453-521

Using TMHMM2.0 algorithm is film to the subcellular compartment of the prediction of AMT polypeptide shown in SEQ ID NO:4.

Embodiment 6: the assay method relevant to the peptide sequence that can be used for implementing the inventive method

AMT polypeptide can cross-film transportation ammonium.Exist and measure the active many assay methods of this class picked-up, comprise the complement test method (Ninneman etc. (1994) EMBO J13:3464-3471) of the yeast strain with endogenous ammonium transporter defect, the picked-up in yeast, xenopus leavis oocytes (Ludewig etc. (2003) J Biol Chem278:45603-45610), vegetable cell, roots of plants (Yuan etc. (2007) Plant Phys143:732-744) and whole strain plant (Hoque etc. (2006) Functional Plant Biology 33:153-163) is measured.This class experimental procedure of measurement AMT activity as well known to those skilled in the art (comprising the AMT activity of AMT polypeptide shown in SEQ ID NO:2).

Embodiment 7: nucleotide sequence shown in clone SEQ ID NO:1

Unless otherwise indicated, otherwise carry out recombinant DNA technology according to the standard method of the following stated: (Sambrook (2001) Molecular Cloning:a laboratory manual, third edition Cold Spring Harbor Laboratory Press, CSH, New York) or Ausubel etc. (1994), Current Protocols in Molecular Biology, the 1st and 2 volumes of Current Protocols.Be described in the Plant Molecular Biology Labfax (1993) of R.D.D.Croy for the standard material of plant molecular engineering and method, by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK) publication.

The synthetic cDNA of the mRNA that uses Phaeodactylum tricornutum from different reproductive stage and different growth conditions to extract is as template, by the encode Arabidopis thaliana cDNA of AMT peptide sequence shown in SEQ ID NO:4 of pcr amplification.In pcr amplification, use the following primer comprising for the AttB site of Gateway restructuring:

1) Prm09458(SEQ ID NO:35, has justice):

5’-ggggacaagtttgtacaaaaaagcaggcttaaacaatgatgcaggccggg-3’

2) Prm09459(SEQ ID NO:36, antisense, complementation):

5’-ggggaccactttgtacaagaaagctgggtacacgagcagcaattaaacc-3′

In standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Also use standard method amplification purifying to there is the desired length PCR fragment of (comprising attB site).Implement subsequently the first step of Gateway method, i.e. BP reaction, " entering clone " that in PCR fragment and pDONR201 plasmid generation body, restructuring is named according to Gateway with generation during this period.Plasmid pDONR201 is as Gateway the part of technology is bought from Invitrogen.

Embodiment 8: the expression vector establishment that uses nucleotide sequence shown in SEQ ID NO:1

In LR reaction, use the clone that enters who comprises SEQ ID NO:3 together with the object carrier transforming for rice subsequently.This carrier contains as functional element on T-DNA border: plant can selective marker; Can enter the Gateway box of recombinating in the object nucleotide sequence generation LR body in clone with intention and described in being cloned in by selection markers expression cassette.Be positioned at the upstream of this Gateway box for the rice GOS2 promotor (SEQ ID NO:34) of constitutive expression.

After LR reconstitution steps, the expression vector pGOS2::AMT (Fig. 4) of generation is converted in agrobacterium strains LBA4044 according to method well-known in the art.

Embodiment 9: Plant Transformation

Rice transforms

The Agrobacterium that contains expression vector is used for transforming rice plant.By the ripe dry seed shelling of the Japanese Cultivar Nipponbare of rice.By hatching in 70% ethanol one minute, subsequently at 2%HgCl ₂in 30 minutes, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The seed of sterilization is containing the upper sprouting of the substratum of 2,4-D (callus inducing medium) subsequently.Hatch in the dark after 4 weeks, embryogenic is cut from scutellary callus and bred on same substratum.After 2 weeks, callus is bred or breeds by subculture on same substratum for other 2 weeks.The subculture 3 days on fresh culture of embryogenic callus sheet, cultivates (to strengthen cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains each body expression vector is independently for common cultivation.Agrobacterium is seeded in to contain on suitable antibiotic AB substratum and at 28 ℃ and cultivates 3.Subsequently bacterium being collected and is resuspended in liquid cultivates in substratum altogether to density (OD ₆₀₀) approximately 1.Suspension is transferred to culture dish subsequently and callus is soaked 15 minutes in this suspension.Callus is organized and on filter paper, is blotted subsequently and be transferred on curing common cultivation substratum and hatch 3 in 25 ℃ in the dark.The callus of cultivating is altogether cultivated 4 weeks under selective agent exists in 28 ℃ in the dark on the substratum that contains 2,4-D.During section, form mushroom resistant calli island at this moment.After this material transfer is hatched to regeneration culture medium and under light, the release of embryo generation potentiality and seedling are in 4-5 week growth subsequently.Seedling cut from callus and hatch 2-3 week at the substratum that contains plant hormone, wherein seedling being transferred to soil from described substratum.The seedling of sclerosis is cultivated under high humidity and short day in greenhouse.

Each construct is produced to approximately 35 independently T0 rice transformant.Primary transformant is transferred to greenhouse from incubator for tissue culture.After copy number at quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only retain performance selective agent tolerance are used for gathering in the crops T1 seed.Seed is gathered in the crops subsequently the 3-5 month after transplanting.Present method produces term single gene seat transformant (Aldemita and Hodges1996, Chan etc. 1993, Hiei etc. 1994) to exceed 50% ratio.

Embodiment 10: phenotype evaluation method

Prepare 10.1 evaluate

Produce about 35 T0 rice transformant independently.Primary transformant is transferred to greenhouse for Growth and yield T1 seed from incubator for tissue culture.Leave 6 events, the T1 offspring of wherein said event separates with 3:1 ratio genetically modified presence/absence.For each event in these events, select to contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.Transgenic plant and corresponding inefficacy zygote are cultivated side by side on random site.Greenhouse experiment is short day (illumination in 12 hours), 28 ℃ and 22 ℃ and relative humidity 70% in the dark under illumination.

4 T1 events T2 from generation to generation according to as do further to evaluate for T1 identical evaluation method from generation to generation, but that each event adopts is more individual.Make plant from sowing time until the ripening stage is passed through digital imagery case for several times.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048x1536 pixel, 1,600 ten thousand colors).

10.2 statistical study: F-check

Use double factor ANOVA (variance analysis) overall evaluation for plant phenotype feature as statistical model.All measuring parameters of whole plants to the whole events with gene transformation of the present invention are implemented F check.Implementing F checks to check gene for the effect of whole transformation events and verifies the mass action (being called again overall gene action) of gene.The threshold value that is used for the significance of true overall gene action is checked and is arranged on 5% probability level for F.Significance F test value indicates gene action, means that not only the difference in phenotype is just caused in existence or the position of gene.

10.3 parameters of measuring

The measurement of biomass correlation parameter

From sowing time until the ripening stage, make plant pass through digital imagery case for several times.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048x1536 pixel, 1,600 ten thousand colors).

The sum that plant shoot divides area (or Leaf biomass) to be different from the pixel of background by counting in the digital picture of dividing from plant shoot is measured.This value averages and changes into by correction the physical surface value of expressing with square millimeter to the picture of taking from different perspectives on same time point.Experiment confirms that the over-ground part plant area of measuring is by this way relevant to the biomass of ground plant part.Over-ground part area is area measured in the time that plant has reached on the time point of its maximum Leaf biomass.Early stage vigor is plant (seedling) area on the ground while sprouting latter 3 weeks.The raising of root biomass is expressed as the raising of total root biomass (being measured as the maximum biomass of the root of observing in plant life); Or be expressed as the raising of root/branch index (ratio of root quality and branch quality in the active growth stage of being measured as root and branch).

The measurement of seed correlation parameter

Ripe primary panicles is gathered in the crops, counted, packs, adds bar code label and in loft drier, is dried 3 at 37 ℃ subsequently.Subsequently by inflorescence threshing and collection and count whole seeds.Use blowing device to separate full grain and empty grain.Discard empty grain and again remainder counted.Full grain is weighed on analytical balance.Full seed number is determined by the full grain number of counting after separating step.The seed gross weight of every strain plant is measured by weigh the whole full grain of gathering in the crops from a strain plant.The capsomere number that every strain plant seed sum is gathered in the crops from plant by counting is measured.Draw thousand seed weight (TKW) according to the full seed number of counting and gross weight extrapolation thereof.Harvest index (HI) is defined as seed gross weight and the ground area (mm of every strain plant in the present invention ²) between ratio be multiplied by again the factor 10 ⁶.The sum of spending of each inflorescence is defined as the ratio between the total and ripe primary panicles number of seed in the present invention.The full rate of seed is defined as in the present invention full seed number and accounts for the total ratio (representing with %) of seed (or little Hua).

Embodiment 11: the phenotype evaluation result of expressing the transgenosis rice plant of the nucleotide sequence of AMT polypeptide shown in coding SEQ ID NO:2

Below show and grown under normal growth condition, expressed the T2 of the transgenosis rice plant of the nucleotide sequence of AMT polypeptide shown in coding SEQ ID NO:2 under the GOS2 promotor control for constitutive expression for evaluation result.

As show as shown in E, comparing with corresponding inefficacy zygote (contrast), there is significant raising in the colored quantity of the early stage vigor of transgenic plant, Aboveground Biomass of Young, root biomass, the seed ultimate production of every strain plant, the full rate of seed, full seed quantity, each inflorescence and harvest index.

Table E: under the control of the GOS2 promotor for constitutive expression, express the T2 of the nucleotide sequence of AMT polypeptide shown in coding SEQ IDNO:2 for the evaluation result of transgenosis rice plant.

Embodiment 12: the example that transforms other crops

Corn transforms

The conversion of corn is according to the modification method of (1996.Nature Biotech14 (6): the 745-50) described methods such as Ishida is carried out.Conversion in corn be that genotype relies on and only specific genotype can be used to and transform and regeneration.Inbred lines A188 (University of Minnesota) or the hybrid using A188 as parent are the good sources of the donor material for transforming, but other genotype also can successfully be used.Mealie is in pollination about 11 days (DAP) results afterwards from maize plant, and now the length of immature embryos is about 1 to 1.2mm.Immature embryos cultivates altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant occur to recover by organ.The embryo cutting on callus inducing medium, cultivate subsequently on corn regeneration culture medium, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or until seedling growth.Green seedling is transferred to maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling of taking root is migrated in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce T1 seed.

Wheat transforms

The conversion of wheat is undertaken by the method that (1996) Nature Biotech14 (6): the 745-50 such as the Ishida such as Ishida describe.Conventionally in conversion, use (can obtain from Mexico CIMMYT) Cultivar Bobwhite.Immature embryos cultivates altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant occur to recover by organ.After hatching with Agrobacterium, embryo on callus inducing medium, extracorporeal culture on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or until seedling growth.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling of taking root is migrated in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce T1 seed.

Transformation of soybean

According to Texas A & M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several business soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, cut hypocotyl, radicle and a slice cotyledon.Further cultivate epicotyl and remaining cotyledon to grow armpit tight knot.These armpit tight knots are cut and hatch with the agrobacterium tumefaciens that contains expression vector.After common cultivation is processed, explant is washed and is transferred to selection substratum.The seedling of regeneration is cut and is placed in seedling elongation medium.The seedling that length is no more than to 1cm is placed on root media until root development.The seedling of taking root is migrated in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce T1 seed.

Semen Brassicae campestris/rape transforms

Use cotyledon petiole and the hypocotyl of 5-6 age in days seedling to transform as the explant for tissue culture and according to (1998, Plant Cell Rep17:183-188) such as Babic.Business Cultivar Westar (Agriculture Canada) is the standard variety for transforming, but also can use other kind.Brassica seed is done to surface sterilization so that external sowing.From external seedling, cut and there is the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with (containing expression vector) Agrobacterium by the cut ends of petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, under illumination in 16 hours, cultivate 2 days.Cultivating altogether after 2 days with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum that contains 3mg/l BAP, cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid (300mg/l) and continues 7, and cultivating containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, until seedling regeneration.In the time that seedling has 5 – 10mm length, seedling is cut and is transferred to seedling elongation medium (containing the MSBAP-0.5 of 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) for root induction.The seedling of taking root is migrated in the soil in greenhouse.From the plant that shows selective agent tolerance and contain single copy T-DNA inset, produce T1 seed.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999Plant Physiol119:839 – 847) to be transformed.The regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains reproducibility plant has been described.For example, any other business alfalfa variety that these reproducibility plants can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and A Atanassov (1985.Plant Cell Tissue Culture4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978Am J Bot65:654-659) in tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol119:839 – 847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant is cultivated altogether 3 days in the dark on the SH inducing culture that contains 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K2SO4 and 100 μ m Syringylethanones.Explant washs in half concentrated Murashige-Skoog substratum (Murashige and Skoog, 1962) and plating is not containing suitable selective agent and suitable microbiotic to restrain on the identical SH inducing culture of Agrobacterium growth containing Syringylethanone.After several weeks, somatic embryo is transferred in the BOi2Y Development culture base that containing growth regulator, does not contain 50g/L sucrose containing microbiotic.Somatic embryo is sprouted subsequently on half concentrated Murashige-Skoog substratum.The sprigging of taking root is cultivated to flowerpot and in greenhouse.From the plant that shows selective agent tolerance and contain single copy T-DNA inset, produce T1 seed.

Cotton Transformation

(upland cotton (Gossypium hirsutum L.) transforms to use agrobacterium tumefaciens on Hypocotyl Explants, to carry out cotton.Commercially available cultivar as Coker130 or Coker312 (SeedCo, Lubbock, TX) be the standard mutation for transforming, but also can use other mutation.By seed-coat sterilizing sprouting darkling.Cut the Hypocotyl Explants of the about 1-1.5 of length centimetre from the seedling sprouting.Hypocotyl Explants is flooded 5 minutes in the agrobacterium tumefaciens inoculum that contains expression vector, then on MS+1.8mg/l KNO3+2% glucose at 24 ℃ co-cultivation approximately 48 hours in the dark.Explant is transferred to and contains suitable bacterium and the same medium of plant selectable marker (renewal several times), until observe the callus of embryogenic., until there is somatic embryo in the cultivation that callus separated and go down to posterity.Make the plantlet maturation on root media from somatic embryo, until grow root.The seedling of taking root is migrated in the potted plant soil in greenhouse.T1 seed is produced by the plant that selective agent is shown to patience and contain single copy T-DNA inset.

Embodiment 13: the example of abiotic stress screening

Arid screening

Under normal culture condition, in basin soil, cultivate the plant from selected event number, until arrive the heading-stage.Then transfer them to " arid " part that stops watering.In the random basin of selecting, insert hygrosensor, to detect soil moisture content (SWC).In the time of the low certain threshold value of SWC, automatically plant is continued to rewater until again reach normal level.Then plant is transferred to normal condition.It is identical with the plant of not cultivating under abiotic stress condition that all the other cultivate (plant maturation, seed are gathered in the crops).Record Growth and yield parameter as cultivated under normal condition described in detail.

Salt stress screening

By coconut fiber and argex(3:1 ratio) culturing plants in the matrix that forms.Use normal nutritive medium at first two weeks of plantlet being transplanted to behind greenhouse.After first two weeks, in nutritive medium, add 25mM salt (NaCl), until results plant.Record Growth and yield parameter as cultivated under normal condition described in detail.

Nutrient (nitrogen) availability reduces screening

In basin soil, cultivate the plant (T2 seed) from 6 events being under normal condition except nutritive medium.All use specific nutritive medium to water to basin from being transplanted to maturation, wherein contain nitrogen (N) content of reduction, conventionally reduce by 7 to 8 times.It is identical with the plant of not cultivating under abiotic stress condition that all the other cultivate (plant maturation, seed are gathered in the crops).Record Growth and yield parameter as cultivated under normal condition described in detail.

Claims (12)

1. the method that improves Correlated Yield Characters compared with control plant in plant, it comprises: the expression that improves the nucleotide sequence of coding ammonium transporter (AMT) polypeptide in plant, and optionally select to have the plant of Correlated Yield Characters of raising, wherein said AMT polypeptide comprises with conserved domain (CD) shown in SEQ ID NO:33 and has at least 50% to increase progressively preferred sequence, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or the structural domain of homoamino acid sequence identity more, shown in wherein said AMT polypeptide and SEQ ID NO:4, AMT polypeptide has at least 40% to increase progressively preferred sequence, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or homoamino acid sequence identity more.

2. according to the process of claim 1 wherein that the nucleotide sequence of described coding AMT polypeptide is the nucleotide sequence shown in SEQ ID NO:3, or can with the sequence of the nucleic acid array hybridizing shown in SEQ ID NO:3.

3. according to the method for aforementioned claim any one, the expression of wherein said raising is by following any or multiple realization: T-DNA activation label, TILLING or homologous recombination.

4. according to the method for aforementioned claim any one, the expression of wherein said raising realizes by the nucleotide sequence of introducing in plant and express coding AMT polypeptide.

5. according to the method for aforementioned claim any one, the Correlated Yield Characters of wherein said raising be following one or more: the root biomass of the early stage vigor of raising, the Aboveground Biomass of Young of raising, raising, every strain plant seed ultimate production of raising, the full rate of seed of raising, the full seed number of raising or the harvest index of raising.

6. according to the method for any one in aforementioned claim, wherein said nucleotide sequence is effectively connected with constitutive promoter, preferably effectively be connected with plant constitutive promoter, more preferably be effectively connected with GOS2 promotor, be most preferably effectively connected with the GOS2 promotor from rice as shown in SEQ ID NO:34.

7. according to the method for any one in aforementioned claim, the nucleotide sequence of wherein said coding AMT polypeptide is from not waiting flagellum door, preferably from Diatomacae, more preferably from Pennales, most preferably from Phaeodactylum tricornutum.

8. construct, it comprises:

(a) nucleotide sequence of coding the claim 1 or 2 AMT polypeptide that define;

(b) one or more control sequences that can drive the nucleotide sequence of (a) to express; With optional

(c) transcription termination sequence.

9. construct according to Claim 8, wherein said control sequence is plant constitutive promoter, preferably GOS2 promotor, more preferably the GOS2 promotor as shown in SEQ ID NO:34.

10. according to Claim 8 or 9 construct is in the purposes for generation of having compared with control plant in the method for plant of Correlated Yield Characters of raising, the Correlated Yield Characters of wherein said raising be following one or more: the root biomass of the early stage vigor of raising, the Aboveground Biomass of Young of raising, raising, every strain plant seed ultimate production of raising, the full rate of seed of raising, the full seed number of raising or the harvest index of raising.

11. for generation of the method for transgenic plant of Correlated Yield Characters compared with control plant with raising, and it comprises:

(i) in plant, plant part or vegetable cell, introduce and express the nucleotide sequence of coding the claim 1 or 2 AMT polypeptide that define, under its control in plant constitutive promoter; With

(ii) under the condition of Promoting plant growth and growth, cultivate described vegetable cell, plant part or plant.

The purposes of the nucleotide sequence of 12. coding claim 1 or 2 AMT polypeptide that defines in raising plant biomass correlated character, the Correlated Yield Characters of described raising comprise following one or more: the root biomass of the early stage vigor of raising, the Aboveground Biomass of Young of raising, raising, every strain plant seed ultimate production of raising, the full rate of seed of raising, the full seed number of raising and the harvest index of raising.

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